Development and Application of a Loop-Mediated Isothermal Amplification (LAMP) Assay for the Detection of Pseudomonas syringae Pathovars pisi and syringae

Round 1

Reviewer 1 Report

This manuscript describes a loop-mediated isothermal amplification (LAMP) assay devised by Kant et al. to differentiate Pseudomonas syringae pv. pisi (Psp) and P. syringae pv. syringae (Pss) in field pea tissues.  The assay appears to have appropriate specificity and sensitivity for application in the field, and is performed with a relatively straightforward protocol.  I am only recommending minor modifications to the manuscript prior to its publication, as detailed below.

General comment: Please italicize all Latin names

Abstract: “Sativum” should be “sativum”

Introduction and lines 40, 43: "PCR reaction" is redundant (= polymerase chain reaction reaction)

Table 1: If the “Field Pea Cultivar” is blank for a given isolate, is it unknown?  Please clarify this in the legend or put “unknown” in the table.

Table 2 and lines 125-127: The accession numbers provided do not yield any results when searched in GenBank. Are they not yet public?

Table 2: An accompanying schematic of the template showing primer annealing sites would be helpful.

How robust is LAMP in the face of random mutations in primer annealing sites?  The authors selected highly conserved sequences, but additional PAML analysis could provide reassurance that the selected sequences might be under purifying selection to maintain that conservation.  Also, SalA is an essential gene, but what about SyrB and AN7?

line 53: Is there actually any annealing happening at 98C?

Please provide references to previous work indicating the degree of correlation between bacterial populations enumerated in overnight leaf exudates versus those counted in homogenized tissues, i.e. how much of the bacteria is released into the exudate?

Table 3: “pathovar” should not be italicized

Section 2.4.3: Were the samples all from the same field?  If so, it is not surprising that a single pathogen was identified.  Based on the scale of this study, the conclusion of “the dominance of Pss as a causal pathogen of BB” (line 283) is unsubstantiated.  The LAMP assay would be a valuable resource for epidemiological surveys of larger areas, which would be required to confirm the prevalence of a given pathogen in the total growing area.

Lines 151-152, 157, and elsewhere: Some VPRI numbers are incorrectly formatted with commas inserted.

Fig 2 legend: The 10 in log10 is generally subscripted.

Line 221: “non” should be “none”

Section 2.2 describes primers designed to detect Pss with primers specific to SalA, but Section 3.2 uses SyrB to detect Pss.  The Discussion also only mentions SalA, despite all of the data being based on SyrB.  Please clarify this discrepancy.

Author Response

Dear Reviewer,

Thank you for your feedback. 

Please see the attachment for the responses we have done in the paper.

Author Response File: Author Response.pdf

Reviewer 2 Report

The overall assessment of the manuscript submitted for review is positive.

I believe that the choice of the journal Agriculture is also appropriate, as the authors present results that can be directly used in agriculture.

The study uses LAMP, a relatively new method of identifying pathogens, which can be carried out in the field, in a very short time and without the need for prior isolation of DNA from the tissue. As shown by the authors, also the origin of the plant material for analysis to distinguish infection by one of the types of Pseudomonas syringae is not important, because virtually any material can be analyzed in a way that allows perfect detection.

The title is appropriate to the content of the work.

The abstract is right. Concise and presenting both the problem and the obtained results, as well as conclusions. The analyzed species is Pisum sativum L. This is the full species name.

Keywords have been omitted.

Apart from minor errors marked in the attached file, such as - no italics or spelling in capital or small letters, I have no remarks on the Introduction.

As for the Materials and methods chapter, I believe that all materials used in the work should be listed in one place. They are included in several tables, but it would be good to list them all together in one chapter in the methodology section. Perhaps even immediately define their purpose for specific analyzes. The work is quite complicated and it would be worth simplifying the task for the reader by immediately presenting the operation scheme in the description of the materials and then in the subsequent stages of presenting the methodology. Similarly, I believe that it should be indicated in another way that unpublished results were used in the research. This information should appear sooner. In general, you can take a look at the methodology and results again - maybe they can be more systematized and organized.

The Results chapter also includes genre names that should be italicized, abbreviations that have not been explained. Similar comments concern the discussion and have been marked in the attached file. Also, not all generic and species names are in italics in the References. 

I recommend minor revision.

Comments for author File: Comments.pdf

Author Response

Dear Reviewer, 

Thank you and we acknowledged the feedback. We have done changes to the manuscript and it has now improved a lot. Please see the attachment with our responses. 

Author Response File: Author Response.pdf