Recombinase Polymerase Amplification Assay for Rapid Field Diagnosis of Stewart’s Wilt of Corn Pathogen Pantoea stewartii subsp. stewartii

Round 1

Reviewer 1 Report

In the manuscript entitled “Recombinase polymerase amplification assay for rapid field diagnosis of Stewart’s Wilt of corn pathogen Pantoea stewartii subsp. stewartii”, the authors apply the diagnostic technique named Recombinase Polymerase Amplification (RPA) coupled with a Lateral Flow Devise (LFD) for rapid and accurate real-time detection of Stewart´s wilt disease. Twelve primers were designed and validated to determine their specificity and sensitivity to detect the pathogen of interest, from other similar and non-similar bacteria. The best performance was observed with CL001-F/R. Due to the results obtained, it is concluded that the technique and designed primers are reliable for their implementation for the detection of Stewart’s Wilt of corn pathogen.

 Additional observations:

Some minor typos: lines 130 (complimentary), 159 (and 10), 376 (roguing).

Images 4B and 4C are not clear enough to observe the lines of control and positive.

Review the manuscript for correction of minor typos

Author Response

All the typographical changes suggested by the reviewer have been incorporated in the revised draft.

Reviewer 2 Report

Authors are request to address few minor comments as stated in the manuscript:

1. The number in figures should be in sequence and corrected

2. Markers in figure2 mast be  denoted in kb.

3. No pictorial evidence of LFA is  observed in manuscript

Comments for author File: Comments.pdf

Author Response

The following changes were incorporated:

1. The call out for Fig. 1 was inadvertently missed in the earlier version. The relevant correction has been made in the revised manuscript.
2. DNA ladder has been properly marked in the revised draft (Fig. 2).
3. LFA: The authors were unable to understand the reviewer's comment. There is no abbreviation as LFA used in the manuscript. The presented data (Fig. 4) is for the LFD (lateral flow dipstick device) based on RPA probes. In addition, the resubmitted version contains a revised and clearer picture of LFD assy (Fig. 4).
4. The faint bands in the PCR (Fig. 2A) are not PCR amplified products, but merely primer dimers.

Reviewer 3 Report

Recombinase Polymerase Amplification Assay for Rapid Field  Diagnosis of Stewart's Wilt of Corn Pathogen Pantoea stewartii  subsp. stewartii

The manuscript describes the development and the optimization of a Recombinase Polymerase Amplification’ (RPA) assay y coupled with a Lateral Flow Device (LFD) for rapid, accurate, and sensitive real-time detection of P. stewartii   subsp. stewartia (pest that causes Stewart's vascular wilt and leaf blight of sweet corn) directly from the infected host offering in-field pathogen detection and timely disease management, for satisfying quarantine and phytosanitary requirements. The authors designed 12 novel primer sets against conserved genomic regions of P. stewartii subsp. stewarti, but only the primers for amplification of intergenic spacer region between capsular polysaccharide genes cpsA and cpsB, determined an unambiguous identification of P. stewartii subsp. stewartii by PCR. Thus, these specific primers were further validated in a simplex RPA assay for specificity against 26 bacterial species representing several Pantoea and other closely related bacterial species/subspecies/strains found in the same niche, and naturally or artificially infected plant samples. The integrated RPA/LFD assay was also optimized for rapid and sensitive on-site detection of P. stewartii  subsp. stewartii with an empirical detection limit of 0.0005 pg/µL of bacterial DNA and 1× 10^2 CFU/mL  (equivalent, for the authors, to 2 bacterial cells) in minimally processed samples.

General comments

The strength of this manuscript is that the authors developed an innovative specific assay, an RPA assay, for the diagnosis of the bacterium Pantoea stewartii subsp. stewartii which is considered a quarantine pest in many countries since it is the causal agent of leaf wilt disease in many host plants, with very high yield losses mainly for sweet corn varieties. This is the first report of an RPA assay developed for the detection of this subspecies of bacterium. The assay is able to differentiate at molecular level Pantoea stewartii  subsp. stewartii from subsp. indologenes which produces less severity in symptom expression when the infection occurs in the host plant. Moreover, the authors significantly validated the assay on bacterial cells and on bacterial genomic DNA extracted from maize leaves and seeds artificially contaminated.

However, the manuscript needs to be implemented and corrected. Below there are some points that should be better clarified.

1)    The English of some sentences should be revised.

2)    The validation to assess the analytical specificity and sensitivity was performed using bacterial cells or genomic DNA from pure culture.  The RPA successfully detected the bacterium. However, an assay for a pathogen should be correctly validated to successfully detect the pathogen in field samples. Generally, the plant matrix/tissue could affect the performance of an assay, especially the molecular assays since most DNA polymerase enzymes can be inactivated or are less performing by due to inhibitors inside the plant tissue. Thus, it would be useful to carry on a validation study using preparations of plant sap recovered from a healthy plant tissue (the same in which the pathogen could be detected in the field) contaminated by bacterial cells or genomic bacterial DNA, at known concentration, also ten-fold diluted to assess the analytical sensitivity. After, these preparations can be processed as the protocol requires. Successively, to complete the validation, diagnostic sensitivity, and specificity would be determined by testing samples from/in the field, or from/in screen-house collections. In this manuscript the assay, RPA, if it would be applied in a monitoring program for the bacterium, was only partially validated and this is a weak point of the manuscript. Moreover, only four field samples were tested using crude sap. Thus, the robustness of RPA assay herein validated to detect the pathogen in field samples is far to be demonstrated.

However, the preliminary results obtained with this innovative assay can be considered promising for the detection of this quarantine pest, thus the manuscript could be accepted but before a relevant revision of the manuscript. Below there are several comments and recommendations.

Specific comments

Abstract

Lane 20: Correct with twelve novel primer sets.

Introduction

This section should be condensed. Some parts are too much descriptive (lanes: 81-99; 116-138). The authors could summarize these parts since the content is reported in detail in the related references. Moreover, the authors could add some reference paper on real-time detection assays published on this pathogen.

Lanes 124-125: correct the sentence. The meaning is not very clear.

Lane 144: The objectives of current research were two-fold: design and….: correct adding a) design and..

 Materials and Methods

Paragraph 2.2.: as reported in the abstract, authors would add that these 12 primer sets are newly designed.

Lane 196: inoculation leaf….: correct with inoculation of leaf...

Paragraph 2.4. lanes 199-208: the authors reported exactly the content of reference 24 (Daher, et al, 2016). The Authors must briefly summarize the content and add the reference. However, the entire paragraph should be summarized in the content and authors could refer to published papers and to the manufacturer’s instruction of the kit.

Lane 225: Correct ‘the nfo target’

Lane 227: correct 10 mins with 10 min

Lane 228: correct the mistake at the beginning of the sentence.

Results

Lanes 237- 243: move the design of the primers in the section of material and methods. Organize better paragraph 3.1 to show only the results.

Pag. 8: in this page, there are figures 2A and 2B; in the text of the manuscript (paragraph 3.1) is only described the figure 2A. There isn’t any description or reference to the results of Figure 2B in the text of the entire manuscript. Authors should add in the text the results shown in Fig 2B.

Table 3: In my opinion, this table could be deleted from the manuscript and added as a supplementary material. The results recovered from table 3 are well described in the text.

Lanes: 274-276: It is not clear if P. stewartii subsp. stewartii strain DC283 was used as negative control and if yes why since in Fig. 2A a positive amplicon was visualized on the gel for this strain.

Legend of figure 3:

-          delete lanes 291-293 from (optimal…channel) and add this content in material and methods and results.

-          Lanes 305-306. Specify more in detail which is shown exactly in Fig. 3D, 3E, and 3F because some data shown in the figures are missed in the legend.

        Lanes 307-310: the authors assert that adequate amplification of RPA assay was observed at 37°C within 20 min in a reaction containing as low 0.0005 pg μL-1 target DNA [TT(F) value =25.8 min] (Fig. 3D).

But the amplification curve produced by the concentration 0.0005 pg/μl (blue curve) is flat as to the signal produced by background and negative controls. Thus, adequate amplification curves were observed until 0.005 pg/μl concentration, which is the detection limit of the assay that can be observed in Fig 3D.

Lanes 314-316: in the text, seedlings are reported in Fig 3E, but Fig. 3 reported infected maize leaf. Please, align text and fig 3E.

Lanse 316-319: the authors assert that the detection limit of RPA assay in desiccated seeds was determined to be 1×10^2 CFU/mL, equivalent to 2 bacterial cells. How did the authors determine that 100 cfu/mL is equivalent to 2 bacterial cells? For other bacteria, 100 CFU/mL corresponds to 100 bacterial cells. The same assertion is reported in the abstract. If it is an error, please correct this information in the text and in the abstract.

Lanes 323-324: [TT(F) value =25.8 min, Fig. 323 3D]…. Delete the content of the brackets. At this point of the manuscript, it makes confusion about which is the related figure.

Lanes 324-326: It is not clear if the DNA extracted from artificially inoculated maize seedlings is the same as reported in the sentence starting at lane 328. In this case, please improve this part of the paragraph.

Figure 4 A and B: The authors would add in both pictures A and B  the detection device with a concentration under the detection limit assessed.

Lane 344-354: Data on standardization and validation were produced using bacterial cells and genomic DNA extraction. To confirm the robustness of RPA on its applicability to detect the pest in field samples, validation data using crude healthy sap artificially contaminated with bacterial cells or bacterial DNA are needed (see general comments point 2). Moreover, only four field samples were tested. Thus, I suggest changing this sentence, deleting the concept of robustness of the assay and referring to these results as preliminary experimental tests.

Discussion

This section should be deeply implemented considering the general comment N. 2 and the previous comment. Authors assert that they validated a reliable, specific, and sensitive RPA assay for the detection of P. Stewartii subsp. stewartii, but they need to specify that the RPA assay was mainly validated (especially the assessment of the detection limit) on bacterial cells or DNA extracted from bacterial cells even if some experiments were also performed on artificially inoculated seeds and leaves.

Lane 362: correct the mistake in the sentence.

Lane 366: correct nonhost with non-host

Lane 382-383: Delete 382-392 and Fig. 1 from Discussion and move this part both in Material and Methods and Results.

Lane 408-409: This conclusion needs to be deeply discussed considering the previous comments on the applicability of RPA assay validated to detect the pathogen in the field. Thus, the section of discussion should be significantly implemented emphasizing that this is the first report on the application of RPA assay for the diagnosis of Pantoea stewartii subsp. stewartii, that the assay can differentiate this subspecies from subsp. indologenes (highlighting why it is important), and that it could be a real-time detection using a portable isothermal nucleic acid detection system or a lateral flow dipstick, that preliminary results are obtained for in-field diagnosis thus further implementation of the assay is urgently required.

 For all the previous comments, I recommend that the manuscript can be accepted with major revisions.

Minor editing

Author Response

Please see the attachment. The authors' response and rebuttals are marked in red font.

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Recombinase Polymerase Amplification Assay for Rapid Field Diagnosis of Stewart's Wilt of Corn Pathogen Pantoea stewartii  subsp. stewartii

I would like to thank the authors to have done all the possible efforts to improve the manuscript with most of the suggested modifications. They greatly improved the text in its understanding and clarity. In particular, they better explained the experiments in fig. 3 D-E-F.

Regarding the general comment 2) and the reply of the authors, the detection of a pathogen in different host plants or different tissues is a critical point that would be evaluated for successful detection of the pathogen due to different inhibitors of the amplification reaction (polysaccharides and phenolic compounds) that can be present in sap recovered from a plant/tissue rather than another [e.g. EPPO standard on Xylella fastidiosa PM 7/24 (5)]. The validation of an assay includes the assessment of different parameters: analytical sensitivity, analytical specificity, repeatability, and reproducibility. The inhibitors can impact some of these, in particular the detection limit of the assay which could be different for each plant matrix.

However, since they better explained the experiments in fig. 3 D-E-F, now it is clear that they validated the assay at least for the matrix ‘desiccated seeds’.

Specific comments

Abstract:

Lanes 27-28: Authors clarified in the responses and in the modified manuscript how they calculated 2 bacterial cells. I suggest to delete 100 cfu/mL and leave only app.  2 bacterial cells or try to explain better the difference, because as it is written could be interpretated 100 cfu/mL=2 bacterial cells which is not correct. You can add app. 2 bacterial cells in the final reaction.

Lane 295: ‘was used the positive control’ correct with ‘was used as positive control’.

Lane 425-426: ‘and performs well with high sensitivity using crude extracts from infected plants and incubated in closed hand under field conditions.’

I do not agree with the high sensitivity of the assay using crude extracts. The authors didn’t assess the detection limit using crude extracts but using purified DNA obtained from bacterial cells or from desiccated seeds by a commercial kit. This purified DNA preparation surely contains fewer inhibitors of the amplification reaction than a plant crude extract. Moreover, even if the assay detected correctly the positive samples from field plants, they analyzed only 4 samples. Thus they can’t know if the sensitivity of the assay is high using crude extracts.

These are preliminary results on a potential future application of this assay in the field, but an extensive validation of the assay using crude extracts should be carried out in the future.

Based on my previous comments, I recommend to accept the manuscript with minor editing.

Comments for author File: Comments.pdf

It'fine. I indicated in the revision report, in specific comments, only one correction

Author Response

Thank you for the very helpful suggestions in improving the manuscript. All the suggested changes have been incorporated. However, the authors reiterate that the assay works well with crude extracts obtained from field-infected plants as well as artificially infected seeds. Even though only a few reactions have been shown in the paper, we have tested more than a hundred samples with accuracy.