Characterization of Tomato Brown Rugose Fruit Virus (ToBRFV) Detected in Czech Republic

Round 1

Reviewer 1 Report

ToBRFV is a epidemic virus worldwidely, and has been caused severe economic loss in Solanaceae crops. This paper reports the identification of ToBRFV infection in Czech Republic by RT-PCR and TEM, and also present the miRNAs of host infected with ToBRFV. 

The structure of the paper should reorganized, the section "TEM" should be following with section "RT-PCR", both results are verifications of identification of virus.

The result of miRNAs should reanalysize, the expressing of miRNAs in infected host by ToBRFV are changed or not?

Author Response

Reviewer 1

ToBRFV is a epidemic virus worldwidely, and has been caused severe economic loss in Solanaceae crops. This paper reports the identification of ToBRFV infection in Czech Republic by RT-PCR and TEM, and also present the miRNAs of host infected with ToBRFV. 

The structure of the paper should reorganized, the section "TEM" should be following with section "RT-PCR", both results are verifications of identification of virus.

Thank you for this comment, the section „TEM“ is following the section RT-PCR.

The result of miRNAs should reanalysize, the expressing of miRNAs in infected host by ToBRFV are changed or not?

Thank you for this comment, the most important information obtained based on small RNA seq, was to obtain the whole genomic sequence of ToBRFV isolate that was detected in the Czech Republic, this was done by small RNA seq. It is not possible to show if the expression of miRNAs was different from „normal“ health condition. Regarding the level of miRNA expression we can only compare the expression of the same ToBRFV isolate in pepper and tomato. Concluding this we used the small RNA sequencing because of: i) to detect ToBRFV; ii) to obtaing whole genome of ToBRFV and iii) to compare the miRNA expression profile in pepper and tomato

Reviewer 2 Report

This manuscript is good enough to be accepted except for minor grammatical errors.  Please read your manuscript carefully and correct any grammatical errors.  The examples given below were found in your abstract.

Line 13: Tomato is the first vegetable consumed in the world.  ----> Tomato is the number one most consumed vegetable in the world.

Line 15: Detection and characterization of this important viral pathogen was ------>Detection and characterization of this important viral pathogen were

Line 19: morphology of the isolate----->the morphology of the isolate

Author Response

Reviewer 2

This manuscript is good enough to be accepted except for minor grammatical errors.  Please read your manuscript carefully and correct any grammatical errors.  The examples given below were found in your abstract.

Line 13: Tomato is the first vegetable consumed in the world.  ----> Tomato is the number one most consumed vegetable in the world.

Thank you very  much for this comment, according to our best knowledge and references lower, tomato should be really the first vegetable consumed in the world. The English was corrected. Please, see references bellow:

Causse, M. et al. (2020). Genomic Designing for Climate-Smart Tomato. In: Kole, C. (eds) Genomic Designing of Climate-Smart Vegetable Crops. Springer, Cham. https://doi.org/10.1007/978-3-319-97415-6_2

Kramer, M.G., Redenbaugh, K. Commercialization of a tomato with an antisense polygalacturonase gene: The FLAVR SAVR™ tomato story. Euphytica 79, 293–297 (1994). https://doi.org/10.1007/BF00022530

Line 15: Detection and characterization of this important viral pathogen was ------>Detection and characterization of this important viral pathogen were

corrected

Line 19: morphology of the isolate----->the morphology of the isolate

corrected

Reviewer 3 Report

The manuscript describes the characterization of tomato brown rugose fruit virus (ToBRFV) isolated from an infected tomato plant from Czech Republic. ToBRFV was mechanically transmitted to Nicotiana, pepper, and tomato plants and assessed for the symptom development and miRNA content. Small RNA sequencing was adopted for the RNAs extracted from the pepper and tomato, assembled complete genome sequences of ToBRFV and performed a phylogenetic analysis with genome sequences of other isolates reported worldwide. Virion structure was observed by TEM.

In my opinion the content of the article doesn’t come under the aim and scope of Diversity Journal. The appropriate content would have been a detailed analysis of the molecular diversity and evolution of the ToBRFV population but that is already published. Please see “Comparative Analysis of Tomato Brown Rugose Fruit Virus Isolates Shows Limited Genetic Diversity. Viruses, 2022 Dec 17;14(12):2816. doi: 10.3390/v14122816”. Therefore, I reject the article for publication journal.

However, the article holds some merits to be published in some other journals that is relevant to its content. While reviewing the manuscript I noted down the shortcomings of the paper and corrections needed. The authors are encouraged to make those corrections to improve the quality of their article.

Suggestions to improve the article:

There was no description about the genome of ToBRFV and a general taxonomy of the virus anywhere in the text. Please include that in the introduction. Also, include a description of the genome regions, explaining the coding and the non-coding region of the two isolates and comparison of the percentage of identity in gene products. More content could be brought into the diversity, phylogeny and evolution of the ToBRFV population.

·       Generally, the manuscript needs a thorough editing for better English for an easy flow in reading.

·                          27: vegetables

·       27: Rephrase the sentence starting with“ Except of-----Middle 29 East [1, 2]”

·       31: “doubtful host [2]”: probable host [2].

·       33: was confirmed in USA (California and Florida)

·       39: Correct the sentence “There is no known resistance to ToBRFV, even a cultivar previously resistant to other tobamoviruses.”

·       48-50: modify to “The seed sequence or seed region in miRNA is represented by a conserved heptametrical sequence. Perfect binding in the seed region has a major impact on the regulatory function of a miRNA.”

·       74: except necrosis of older leaves

·       92: “with a tap water” remove ‘a’

·       98: “Nicotiana benthamiana plants stopped the growth and chlorotic.” modify sentence

·       99: and became chlorotic

·       Results and materials and methods are mixed up in 2.1.2 Move the symptoms on the inoculated plants to Result section and indicate the figure # in the respective places. Do you have figures on the indicator plants?

·       156: “The library for small RNA sequencing was used the same as for real time RT-PCR detection- Did not understand the sentence, please rephrase it.

·       182-184: What program was used for the phylogenetic analysis?

“Phylogenetic analysis was applied using these parameters; Three method: Fast minimum Evolution, Max Seq Difference: 0.75 (ncbi.nlm.nih.gov/blast/treeview), 50 the most similar ToBRFV genomic sequences were included.”- Please modify the sentence to make things clear for the reader.

·       198: “bioassay was finished” to bioassay was completed

·       218-221: Modify the sentence to something like “In phylogenetic analysis the isolates clustered with tomato isolates reported from UK and Italy (UK isolates 2020015323_A [Acc. No. OM515231], 2020015323_B [Acc. No. OM515232] Italy isolate Tom-BA21 [OK624678]).

·       240: Based on the results (subchapters 3.2.1 and 3.2.2): There is no 3.2.2 section in the manuscript

·       Complete genome sequences of two isolates were obtained in the study but no comparative analysis on those two sequences other than the phylogenic tree. Were these two isolates 100% identical? The genome of the ToBRFV has to be described in terms of the coding and noncoding regions and include a genome organization figure.

·       “Based on the results (subchapters 3.2.1 and 3.2.2), the newly described Czech ToBRFV isolate inoculated 2 times – on pepper (PP1) and on tomato (TT2) – was evaluated as identical at the level of genomic sequence.”

The information provided in 3.2.1 is insufficient to make this statement. There are no comparison of the sequences of the isolates from tomato compared either at nucleotide and amino acid level. Please include that.

·       236 Correct Figure 3 legend: “sequences of PP1/TT2 isolates”

·       246-248: what is the relevance of this information?

·       Ct values are too low in qRT-PCR due to too much starting virus RNA. Too much starting template may inhibit an assay especially the sensitive assays like qPCR.

Author Response

Reviewer 3

The manuscript describes the characterization of tomato brown rugose fruit virus (ToBRFV) isolated from an infected tomato plant from Czech Republic. ToBRFV was mechanically transmitted to Nicotiana, pepper, and tomato plants and assessed for the symptom development and miRNA content. Small RNA sequencing was adopted for the RNAs extracted from the pepper and tomato, assembled complete genome sequences of ToBRFV and performed a phylogenetic analysis with genome sequences of other isolates reported worldwide. Virion structure was observed by TEM. In my opinion the content of the article doesn’t come under the aim and scope of Diversity Journal. The appropriate content would have been a detailed analysis of the molecular diversity and evolution of the ToBRFV population but that is already published. Please see “Comparative Analysis of Tomato Brown Rugose Fruit Virus Isolates Shows Limited Genetic Diversity. Viruses, 2022 Dec 17;14(12):2816. doi: 10.3390/v14122816”. Therefore, I reject the article for publication journal. However, the article holds some merits to be published in some other journals that is relevant to its content. While reviewing the manuscript I noted down the shortcomings of the paper and corrections needed. The authors are encouraged to make those corrections to improve the quality of their article.

Suggestions to improve the article:

There was no description about the genome of ToBRFV and a general taxonomy of the virus anywhere in the text. Please include that in the introduction. Also, include a description of the genome regions, explaining the coding and the non-coding region of the two isolates and comparison of the percentage of identity in gene products. More content could be brought into the diversity, phylogeny and evolution of the ToBRFV population.

Thank you for this comment, we added the information about the genome structure, genomic regions, and description of coding and non-coding regions. We also improved the description of the phylogenetic part. The obtained isolates were identical.

Generally, the manuscript needs a thorough editing for better English for an easy flow in reading.

27: vegetables

Corrected

27: Rephrase the sentence starting with“ Except of-----Middle 29 East [1, 2]”

Corrected

31: “doubtful host [2]”: probable host [2].

Corrected

33: was confirmed in USA (California and Florida)

Corrected

39: Correct the sentence “There is no known resistance to ToBRFV, even a cultivar previously resistant to other tobamoviruses.”

Corrected

48-50: modify to “The seed sequence or seed region in miRNA is represented by a conserved heptametrical sequence. Perfect binding in the seed region has a major impact on the regulatory function of a miRNA.”

Corrected

74: except necrosis of older leaves

Corrected

92: “with a tap water” remove ‘a’

Corrected

98: “Nicotiana benthamiana plants stopped the growth and chlorotic.” modify sentence

Corrected

99: and became chlorotic

Corrected

Results and materials and methods are mixed up in 2.1.2 Move the symptoms on the inoculated plants to Result section and indicate the figure # in the respective places. Do you have figures on the indicator plants?

Description of the symptoms was moved according to the comment. We also added supplementary photo of Nicotiana benthamiana.

156: “The library for small RNA sequencing was used the same as for real time RT-PCR detection- Did not understand the sentence, please rephrase it.

Corrected

182-184: What program was used for the phylogenetic analysis?

The Phylogenetic analysis was applied using these following parameters:; Three method: Fast minimum Evolution, Max Seq Difference: 0.75 (ncbi.nlm.nih.gov/blast/treeview), 50 the most similar ToBRFV genomic sequences were included.

The sentence was modified according to the comment.

198: “bioassay was finished” to bioassay was completed

Corrected

218-221: Modify the sentence to something like “In phylogenetic analysis the isolates clustered with tomato isolates reported from UK and Italy (UK isolates 2020015323_A [Acc. No. OM515231], 2020015323_B [Acc. No. OM515232] Italy isolate Tom-BA21 [OK624678]).

The sentence was corrected

• 240: Based on the results (subchapters 3.2.1 and 3.2.2): There is no 3.2.2 section in the manuscript

The numbering of 3.2.3 was changed to 3.2.2, there was a mistake

• Complete genome sequences of two isolates were obtained in the study but no comparative analysis on those two sequences other than the phylogenic tree. Were these two isolates 100% identical? The genome of the ToBRFV has to be described in terms of the coding and noncoding regions and include a genome organization figure.

Thank you for this comment, we added the mentioned information.

• “Based on the results (subchapters 3.2.1 and 3.2.2), the newly described Czech ToBRFV isolate inoculated 2 times – on pepper (PP1) and on tomato (TT2) – was evaluated as identical at the level of genomic sequence.”

The information provided in 3.2.1 is insufficient to make this statement. There are no comparison of the sequences of the isolates from tomato compared either at nucleotide and amino acid level. Please include that.

Thank you for this comment, we added more information supporting the statement.

• 236 Correct Figure 3 legend: “sequences of PP1/TT2 isolates”

Thank you, corrected

• 246-248: what is the relevance of this information?

This information have to be included how deep sequncing was provided by SBS and how was the distribution between the 2 samples.

• Ct values are too low in qRT-PCR due to too much starting virus RNA. Too much starting template may inhibit an assay especially the sensitive assays like qPCR.

Thank you for this comment. This assay was done in certified laboratory: Division of Plant Pests Diagnostics, National Reference Laboratory, Central Institute for Supervising and Testing in Agriculture, Šlechtitelů 773/23, 779 00 Olomouc, Czech Republic

This is the highest authority in the Czech Republic, so we have to respect the results from this governamental lab. We used the RNA samples provided by this lab and these samples were clearly positive, we will discuss the level of sensitivity for their next testing runs.

Reviewer 4 Report

1). Manuscript ID: Diversity-2097710

2). Manuscript Title: Characterization of tomato brown rugose fruit virus (ToBRFV) detected in Czech Republic

3). Please modify the manuscript based on the following comments.

--Add scientific authority at the end of binomial names of all species when they are mentioned for the first time in the manuscript.

--Include full forms of all abbreviations/acronyms mentioned in the manuscript.

--Extensive editing of English language and style required.

--Rewrite the following sentences: Lines 27, 28, 39, 40, 71 to 74, 98 to 112, and 182 to 184

--Please include symptoms on inoculated plants information in the ‘Results section’ instead of Materials and Methods.

--Figure 3: Include information about how the confidence level for the dendrogram was determined.

Author Response

Reviewer 3

1). Manuscript ID: Diversity-2097710

2). Manuscript Title: Characterization of tomato brown rugose fruit virus (ToBRFV) detected in Czech Republic

3). Please modify the manuscript based on the following comments.

--Add scientific authority at the end of binomial names of all species when they are mentioned for the first time in the manuscript.

Thank you for this comment, the text was corrected according to this comment.

--Include full forms of all abbreviations/acronyms mentioned in the manuscript.

Thank you for this comment, the text was corrected according to this comment.

--Extensive editing of English language and style required.

Thank you for this comment, we used a service provided by native speaker

--Rewrite the following sentences: Lines 27, 28, 39, 40, 71 to 74, 98 to 112, and 182 to 184

Corrected

--Please include symptoms on inoculated plants information in the ‘Results section’ instead of Materials and Methods.

Thank you for this recommendation, this change is logic and very fruitful to keep the correct flow of the text. The text was transferred according to this comment.

--Figure 3: Include information about how the confidence level for the dendrogram was determined.

We included these parameters into the legend of the phylogenetic tree (Figure 3). Thank you this comment.

Round 2

Reviewer 4 Report

1). Manuscript ID: Diversity-2097710

2). Manuscript Title: Characterization of tomato brown rugose fruit virus (ToBRFV) detected in Czech Republic

Thank you for revising the manuscript according to the suggestions.