Phylogenetic Study of Alternaria Potato and Tomato Pathogens in Russia

Round 1

Reviewer 1 Report

Comments for author File: Comments.docx

Author Response

Dear editor and reviewer, we would like to thank the reviewer for careful reading of this manuscript and for constructive suggestions. Our response follows. The corrections are marked with blue in the text. Abstract Line 14: There is no references in abstract. The references should only be in the other sections. Abstract is a resume of the work, and all the information is more detailed in the other sections of the work where it is the respective reference. Accept, we deleted it Introduction Line 26: It would be useful the addition of more information to justify the importance of these crops. We added corresponding sentence: The volume of potato production in Russia is about 30 million tons and financial losses from the development of potato diseases can be very significant. 2. Material and Methods 2.1 Isolates 3) It is possible to distinguish the places of tomato and potato collection? I think that the addition of this work will be better. In order not to make table 1 enormously big we indicated collection sites under the figure 1 2.2. PCR and sequencing 2.3. Phylogenetic analysis 3. Results 3.1. Phylogeny 4) Line 108: There something in bibliography that can help to justify the use of A. nitrimali species as a suitable outgroup? The association of this information with a reference would be better. There is no consensus in this point. Actually, we tried other species and topology of the trees were the same. So we decided to use species isolated from Solanaceae plant. Here are some examples of outgroup species used in my references: 1) Alternaria calendulae in Gannibal, P. B., Orina, A. S., Mironenko, N. V, & Levitin, M. M. (2014). Differentiation of the closely related species, Alternaria solani and A. tomatophila, by molecular and morphological features and aggressiveness. European Journal of Plant Pathology, 139(3), 609623. https://doi.org/10.1007/s10658-014-0417-6 2) A. alternata in Rodrigues, T. T. M. S. (2009). Molecular and morphological characterization, with inference about recombination, for Alternaria species related to early blight of potato and tomato, Doutorado, 91. 3) A. mimicula in Peixoto, C. C., Cabral, C. S., Fonseca, M. E. N., Boiteux, L. S., & Reis, A. (2021). Species diversity, novel interactions and absence of well-supported host-guided phylogenetic groupings of Neotropical Alternaria isolates causing foliar lesions in Solanaceae. Journal of Applied Microbiology, 131(5), 24662487. https://doi.org/10.1111/JAM.15115 5) Line 129: The name of the species should be italic. The correction has been made. 4. Discussion 6) Line 146: it is better to change the apparently word for another that can transmit more certainty about your discovery. As it was the first record it is important to highlight that information. Accept, the text has been revised as suggested: “In the present work, we first discovered a strain of this species on potato. We confirmed disease caused by A. alternaricida on potato plants. The corresponding sentences are marked with grey. 7) Line 148: In the information about the species associated with tomato, add an also, because it is two species already associated with the potato samples, and this way, it makes the information more continuous. Accept, the text has been revised as suggested: 8) Line 154: Do you have any reference number for the DNA sequence? I know that information takes time, but if there is any reference for this, it would be better. If not, do not worry, it can be associated later. We indicated genbank accession numbers in the text.

Author Response File: Author Response.doc

Reviewer 2 Report

I would recommend to publish this work as a short communication as, in my opinion, the results are not enough for an article. I would recommend to summarize this publication (make it brief and concise) and submit it as a short communication/report.

Please add a paragraph in M&M of bioinformatic analysis to describe the different steps you carried out.

The presentation of the results must be improved. Some of bioinformatic results appear among the phylogeny subtitle (the only subtitle in the results section).

The M&M section also must be improved.

Line 68: Which PCR program did you use ? Please add it in the supplementary files

Line 76: Which version of Geneious did you use ?

 

Good Luck!

Sincerely,

 

Author Response

Dear editor and reviewer, we would like to thank the reviewer for careful reading of this manuscript and for constructive suggestions. Our response follows. The corrections are marked with yellow in the text. I would recommend to publish this work as a short communication as, in my opinion, the results are not enough for an article. I would recommend to summarize this publication (make it brief and concise) and submit it as a short communication/report. It first started as a quite compact manuscript, and all of the suggestions resulted in its current volume. Moreover, this type of manuscript cannot be published without tables and figures, which makes it bigger than a short report. This work is dedicated to the diversity of pathogenic species, and suit perfectly to the journals topic. Please add a paragraph in M&M of bioinformatic analysis to describe the different steps you carried out. We added corresponding paragraph: «2.3.1. Bioinformatic analysis Sequences were aligned with the MAFFT version 7 web tool (http://mafft.cbrc.jp/alignment/server/) with subsequent manual processing. Phylogenetic reconstructions were performed with maximum likelihood (ML) and Bayesian (BI) analyses. Nucleotide substitution models for BI were chosen with TOPALI v. 2.5 based on the Bayesian information criterion (BIC). Bayesian analyses were performed with Geneious v. 7.13. In these analyses three parallel runs with four chains each and other default parameters were run for one million generations. A burn-in of 25 % was used in the final analyses, ensuring the average standard deviation of split frequencies had reached < 0.01 for all data sets. Support at nodes was indicated when posterior probabilities were ≥ 0.6. For ML analyses, the best-fit substitution model for the alignment was estimated based on the Akaike information criterion (AIC) using the IQ-TREE Web Service (http://iqtree.cibiv.univie.ac.at/). The Tamura-Nei (TN) [11] model plus empirical base frequencies allowing for a proportion of invariable sites was chosen for the "potato" dataset. For the "tomato" dataset, TN plus empirical base frequencies and a freeRate model with 2 categories were used. The RAxML program ver. 7.0.3 was used for the heuristic search.» The presentation of the results must be improved. Some of bioinformatic results appear among the phylogeny subtitle (the only subtitle in the results section). We added « 3.1. Bioinformatic analysis » paragraph in results. The M&M section also must be improved. Line 68: Which PCR program did you use? Please add it in the supplementary files We added corresponding sentences to the MM paragraph «The PCR program consisted of an initial denaturing step at 94°C for 5 min, 35 amplification cycles, and an additional extending step at 72°C for 3 min. For the primer pairs ITS1/ITS4, RPB2-5F2/ fRPB2-7cR, EF1-728F/ EF1-986R, and Alt-for / Alt-rev, the amplification cycles were 94°C for 30 s, 55°C for 30 s, and 72°C for 30 s. For the primer pair gpd1/ gpd2, the amplification cycles were 94°C for 30 s, 62°C for 30 s, and 72°C for 1 min.» Line 76: Which version of Geneious did you use? The correction has been made; we indicated the version in text

Author Response File: Author Response.doc

Reviewer 3 Report

Phylogenetic study of Alternaria potato and tomato pathogens in Russia

The authors traveled to many places, collected, and isolated Alternaria from invested potato and tomato and looked to see which taxa were causing the Early blight. Taxa ID was completed using multigene phylogeny. The manuscript is straightforward and scientifically sound. There is one major issue and some line items that should be addressed.

 

Major issue is that the authors have completed a Bayesian inference in addition to the ML tree and there is no mention of how this was done in the methods. This is a phylogenetic study so it's important to describe and show the outcome of both phylogenetic reconstruction methods.

Minor issue associated with the major issue: They state that the topologies were similar with relative support values (line 105) however there is no indication of this. They need to state either unpublished or add this information to the methods and add thickened branches to the phylogenetic trees indicating Bayesian posterior probabilities greater than or equal to 95% as do other authors.

Line-item issues:

Abstract:

Lines 16-17: A. solani was duplicated in the list of five pathogens. One of the A. solani needs to be changed to A. protenta. Also consider listing the species in alphabetical order.

 

Materials and Methods: Please add the Bayesian inference methods. This is preferable to leaving it out as it makes the manuscript support stronger.

Line 64: rDNA is awkward as the first word in a sentence. If possible, re-order or rewrite the sentence.

 

Table 2: The column Species would be clearer to the reader if it was changed to Current species name.

 

Line 110: Consider revising: Isolates from affected potato leaves…

First part of the sentence is confusing. Also, consider listing the species in alphabetical order.

 

Discussion: Please adjust line 146-148: As written, these sentences seem unsure, so if the authors found no literature after a review. Just make a definitive statement that this research provides the first observation that A. alternaricida can cause disease in potato plants.

 

Table 1. Isolates used in this study. The alt is not consistent with its previous name: alt a1. Please adjust for consistency.

Author Response

Dear editor and reviewer, we would like to thank the reviewer for careful reading of this manuscript and for constructive suggestions. Our response follows. The corrections are marked with green in the text. Major issue is that the authors have completed a Bayesian inference in addition to the ML tree and there is no mention of how this was done in the methods. This is a phylogenetic study so it's important to describe and show the outcome of both phylogenetic reconstruction methods. The correction has been made. We added corresponding sentences: Nucleotide substitution models for BI were chosen with TOPALI v. 2.5 based on the Bayesian information criterion (BIC). Bayesian analyses were performed with Geneious v. 7.13. In these analyses three parallel runs with four chains each and other default parameters were run for one million generations. A burn-in of 25 % was used in the final analyses, ensuring the average standard deviation of split frequencies had reached < 0.01 for all data sets. Support at nodes was indicated when posterior probabilities were ≥ 0.6. Minor issue associated with the major issue: They state that the topologies were similar with relative support values (line 105) however there is no indication of this. They need to state either unpublished or add this information to the methods and add thickened branches to the phylogenetic trees indicating Bayesian posterior probabilities greater than or equal to 95% as do other authors. The correction has been made. We indicated both supports at nodes. Abstract: Lines 16-17: A. solani was duplicated in the list of five pathogens. One of the A. solani needs to be changed to A. protenta. Also consider listing the species in alphabetical order. The correction has been made Materials and Methods: Please add the Bayesian inference methods. This is preferable to leaving it out as it makes the manuscript support stronger. The correction has been made. We added corresponding sentences: Nucleotide substitution models for BI were chosen with TOPALI v. 2.5 based on the Bayesian information criterion (BIC). Bayesian analyses were performed with Geneious v. 7.13. In these analyses three parallel runs with four chains each and other default parameters were run for one million generations. A burn-in of 25 % was used in the final analyses, ensuring the average standard deviation of split frequencies had reached < 0.01 for all data sets. Support at nodes was indicated when posterior probabilities were ≥ 0.6. Line 64: rDNA is awkward as the first word in a sentence. If possible, re-order or rewrite the sentence. Accept, the text has been revised as suggested: «The internal transcribed spacer 1 (ITS1) and ITS2 regions and the 5.8S ribosomal DNA (rDNA) region of the fungi were amplified with ITS1 and ITS4 primers » Table 2: The column Species would be clearer to the reader if it was changed to Current species name. The correction has been made Line 110: Consider revising: Isolates from affected potato leaves First part of the sentence is confusing. Also, consider listing the species in alphabetical order. Accept, the text has been revised as suggested: « Isolates from affected potato leaves included species of Alternaria alternariacida, A. grandis, A. linariae, A. protenta, A. solani (Fig. 2, Tabl. 1).» Discussion: Please adjust line 146-148: As written, these sentences seem unsure, so if the authors found no literature after a review. Just make a definitive statement that this research provides the first observation that A. alternaricida can cause disease in potato plants. Accept, the text has been revised as suggested: In the present work, we first discovered a strain of this species on potato. We confirmed disease caused by A. alternaricida on potato plants. The corresponding sentences are marked with grey. Table 1. Isolates used in this study. The alt is not consistent with its previous name: alt a1. Please adjust for consistency. The correction has been made

Author Response File: Author Response.doc

Round 2

Reviewer 2 Report

Dear authors,

Thank you for your answers and corrections. 

I think it would be better to a conclusion part in which you summarise in 2 words the findings of your work and giving some perspectives.

 

Sincerely,

Author Response

Dear editor and reviewer,

Thank you for your patience and advices. 

The authors are appreciated for your comments and suggestions on the manuscript.

The corrections are marked with green in the text.

We added corresponding sentences: Thus, we found five pathogenic Alternaria species on potato and two species on tomato plants in Russia. These findings allow us to study the host range and possible options for disease control.

Author Response File: Author Response.doc

Reviewer 3 Report

The authors have completed the requested changes. The only current suggestion is that the author revise the statement in Figure 2 and Figure 3 to reflect that both  branch values indicate ML/Bayesian support.  Currently, they only state ML. 

Author Response

Dear editor and reviewer,

Thank you for your patience and advices. 

The authors are appreciated for your comments and suggestions on the manuscript.

The corrections are marked with yellow in the text.

We added corresponding sentences: "Bayesian posterior probabilities followed by ML bootstrap values are shown at nodes." under both of the figures

Author Response File: Author Response.doc