Molecular Diagnostics in Tomato: Chip Digital PCR Assays Targeted to Identify and Quantify Clavibacter michiganensis subsp. michiganensis and Ralstonia solanacearum in planta

Round 1

Reviewer 1 Report

This is a manuscript with innovative and very useful results, since both bacteria are a global phytosanitary issue.

The authors present an accurate diagnosis with the digital chip

Comments for author File: Comments.pdf

Author Response

Many thanks for the appreciation to our work. All the corrections suggested have been done in the revised version of the manuscript.

Reviewer 2 Report

Authers are proposing this technique over the real time PCR camparative analysis, by authers or supprted studies are missing in disscuisson.

Figures 1-5, all of them need to be modified in easy to read form, numericals on X-Y axis are impossible to read also not mensioned in figure legends.

Author Response

Authors are proposing this technique over the real time PCR comparative analysis, but authors or supported studies are missing in discussion.

Many thanks for this comment. We have implemented the suggested part in the Discussion section.

Figures 1-5, all of them need to be modified in easy to read form, numericals on X-Y axis are impossible to read also not mentioned in figure legends.

We agree with your criticism. The readability of the figures has been improved, both in terms of better resolution of the images and greater clarity of the captions.

Reviewer 3 Report

This study presents diagnostic assays for detection of Clavibacter michiganensis and Ralstonia solanacearum, showing their work using purified DNA as well as DNA isolated from xylem sap of infected tomatoes.

The study appears to be well conducted, presented and organized, however many key details are missing from the methods section, and other limitations of the study are unacknowledged.  For example, in section 2.1, there is insufficient information on the sources of DNA used in the study.  The method details must be included for purification, kits used, strains used, culture conditions etc. 

Which specific isolates used is of great importance to the claims of broad applicability, especially using the Rs 16S primer/probe set.  It appears that only a single isolate was tested, similar to the testing by Dreo et al.  Thus, the authors rely on the more broad testing of Weller et al. (from more than 20 years ago - before phylotype designations were developed).  However, even in the Weller dataset, the 16S primer/probe set did not give positive results for all Ralstonia solanacearum isolates.  Thus it is important to discuss these limitations with regard to the conclusion of this manuscript.

All of the cdPCR plots (Figures 1-5) are provided in low resolution so that axis labels/values are unreadable.  Please provide in higher resolution.

Other minor comments:

Lines 63-66 – This sentence is awkward and unclear

End of introduction – dPCR and cdPCR need to be defined in the body of the paper at first use (in the abstract is not sufficient)

Sections 2.1, 2.2, 2.3 – Qubit kit/reagents used should be specified.

Figure 3 caption – “blank”

Line 303 – “serological”

Author Response

Please see the attachment.

Author Response File: Author Response.docx

Reviewer 4 Report

The manuscript describes the development of two test systems for the dPCR detection of two bacterial pathogens, Rs and Cmm, causing significant losses of tomato yield. In general, the manuscript is well-written, though, in my opinion, Discussion part could be slighty shortened. Instead, it would be good to add some discussion of practical benefits of the developed test system. As I know, equipment for the dPCR is quite expensive.

In any case, the paper can be accepted for publication; I have only one comment (see below):

Line 176-177: authors wrote about Rs and Cmm isolates collected from different regions. However, no any mentioning of these samples and their isolation and cultivation is shown in the Materials and Methods section.

Author Response

The manuscript describes the development of two test systems for the dPCR detection of two bacterial pathogens, Rs and Cmm, causing significant losses of tomato yield. In general, the manuscript is well-written, though, in my opinion, Discussion part could be slighty shortened. Instead, it would be good to add some discussion of practical benefits of the developed test system. As I know, equipment for the dPCR is quite expensive.

Many thanks for your suggestion. The “generic” part of Discussion has been shortened, whereas the practical benefits of the assays developed in dPCR format have been presented and discussed.

In any case, the paper can be accepted for publication; I have only one comment (see below):

Line 176-177: authors wrote about Rs and Cmm isolates collected from different regions. However, no any mentioning of these samples and their isolation and cultivation is shown in the Materials and Methods section.

Many thanks for this comment, that is even shared by another reviewer. We have implemented the section 2.1 including a new table (Table 1) summarizing the methodological details about the bacterial strains used.

Round 2

Reviewer 3 Report

The manuscript is improved and major concerns have been resolved.

The figure resolution is much better now, but it would still be desirable to have the numeric axis labels of all the scatter plots readable, which they still are not.

Addition of the strain/source table in section 2.1 is very good.  I would recommend addition of footnotes to this table to specify the abbreviations used (strain names and culture media names).

Author Response

Dear Reviewer, many thanks for your comments and help.

First of all we added the legend to Table 1, which was completely missing in the previous version of the manuscript, due to our gross mistake.
Regarding the definition of the figures, this in the word version of the manuscript is good, but in the pdf version it is bad. We therefore enclose the single figures asking for help from the editorial staff to improve the final pdf.