Establishment of a Serology- and Molecular-Combined Detection System for Youcai Mosaic Virus and Its Application in Various Host Plants
Round 1
Reviewer 1 Report
Comments and Suggestions for Authors
The manuscript of Feng et al. describes some methods to detect the YoMV. However, several missing information in the methodology, such as confirmation of the viral isolate, and other experimental parameters, including statistical analysis, and cros-reactivity assays, were not provided. These are the main flaws of the work that cast in doubt on the sensitivty and specifity of theses methods to detect YoMV. Based on my comments, major revisions are necessary.
Major comments
Introduction
L44-46: This sentence is confusing. What the diference of YoMV x TMV-cg x CRMV x ORMV? Are they genetically suficiente to separate into species? If no, maybe the serological techniques are not specific to YoMV. No reference was added. Please explain.
Materials and Methods
The viral isolate is missing information such as original host, year of collection, and locality. Was YoMV molecularly confirmed before to be used in the assays?
Methodology parameters are also missing. How many samples were tested to confirm the efficiency of each test (Wetern Blotting, Immuno- Dot Bloting, ELISA, RT-PCR, S-RT-LAMP)? Were antibody and/or samples dilued (e.g., 1:10, 1:100, and 1:1,000) to confirm the efficiency? Were used positive and negative controls? Were used plants plants of what species, age, symptomatic or not?
Have cross-reactivity assays been conducted with other tobamovirus? this will confirm the specifiicty of the antibody.
Results
Some information is a repeat of the methodology section and others must be included in the methodology section (see comments above, see L165-175; L239-244; L272-275; L296-299; L320-322, L345-354). Please, rewrite this section to keep only the results.
Minor comments
L36-42: It is important to describe the genus of each viral species.
L161: Table 2 was not provided.
L187-189; 218-221: Move to discussion section.
L239: “it was” instead “we”
L302-302: If statistical analysis was used, its must be fisrt described in the respective methodology section.
L390-395: If infection was experimentally observed, these plantas have no impact on the epidemiology of the disease.
L403-408, L434: Supplementary figures were not provided.
L445-446: What experiments? Please explain
Author Response
Please see the attachment.
Author Response File: 
Author Response.docx
Reviewer 2 Report
Comments and Suggestions for Authors
This present manuscript describes the preparation of antisera against YoMV with the objective of developing serological-based assays such as ELISA, Western blot, dot-blot, and S-RT-LAMP.
Suggestions were made to improve the manuscript.
Line 33 – A number of symptoms affecting the foliage are described, which are associated with the infection by several viruses. However, in the following sentence, the authors attribute the symptoms to specific viruses affecting radish plants. Please begin by providing the names of the viruses and then the symptoms they cause.
Line 57 – Please confirm whether the detection method used by Cai et al. was RT-PCR or PCR?
Line 59 – Please provide a full description of the abbreviation ReMV.
Line 88 – It is unclear whether the primers used to construct the recombinant expression vector are described in section 2.1.
Line 119 – Please clarify the meaning of p.12?
Line 120 – For western blotting, which plant sample was utilized? And which parts of the plant were used? The SDS-PAGE loading buffer is not typically used for protein extraction. Please amend accordingly.
Line 130 – The reference to figures should be confined to the Results section and excluded from the Materials and Methods section. See also line 139.
Line 131 – The explanation of ELISA procedure needs further description. The preparation of plant extracts is not a common practice. Please provide a detailed description of the composition of the Protein extraction buffer. Please provide a description of the volume of antibody used in each well of the ELISA plate.
Line 165 – It would be beneficial to the discussion about the differences between YoMV-YZ and YoMV-GD if the putative amino acid sequences of these two isolates could be provided.
Line 187 – Figure 1D is missing.
Line 208 – Please provide the concentration value of the recombinant protein.
Line 242 – The authors state that RT-PCR was performed, yet the Materials and Methods section does not include a description of the RT-PCR protocol.
Line 246, Line 251 – The description of figure 3 in the text does not align is not with the legends in the figure. Additionally, it is unclear which panel of the figure shows the results of the Western blot.
Line 277, 278 – Figure 4 is missing in the manuscript.
Line 384 – The information about the genetic variability of YoMV and the explanation about the choice of YoMV-YZ for this study should be provided in the Introduction section.
In supplementary Fig.1, please highlight the isolates YoMV GD CP and YoMV-YZ in bold, if feasible, as the red triangular arrow is not visible.
Comments on the Quality of English Language
The manuscript is well written and easy to understand.
Author Response
Please see the attachment.
Author Response File: 
Author Response.pdf
Round 2
Reviewer 1 Report
Comments and Suggestions for Authors
No comments.
