Genetic Diversity and Pathogenicity of Phytophthora infestans Isolates on Four Solanum tuberosum (Potato) Cultivars in Nariño, Colombia

Round 1

Reviewer 1 Report

The work gave an insight in to the genetic evolution of Phytophthora infestans isolates on different cultivars of Solanum tuberosum (Potato). The findings from the work  contributed immensely to the field of evolutionary genetics and science at large.

The work highlight the  novel approach to breeding and how to  managing disease in potato crops so as to increase more yield, by rotating crops and promising cultivars could be useful  to maintain sustainable crop viability against evolving pathogenic threats.

1. In ine 32-34 where potato was cited as the most consumed in Columbia, its a nice statement but no author was cited ,the statement cant be blank.

2. Under materials and methods line 127 the authors said they used three (3) potato cultivars, while in line 11 of the abstract part they mentioned four (4) cultivars were used in the study. Please the authors should check effect the correction.

3. In line 134 the sporangia/ml should be sporangia/mL

4. In the results part, Figure 1a & b the grid lines should be removed. The x and y axis lines should seen clearly and black to show the lines to the readers. Furthermore, points in both the y and x axis need to format the axis and tick marks outside as seen in figure 2.

5. In figure 1b, by right hand side of the figure, the listed isolates should be ''isolates'' not isolated as it appeared

6. All Phytophthora infestans in the text need to be italized as a scientific name. Those not italized were detected in lines 10,78,87 and 177 to mention a few.

Author Response

Please see the attachment. 

Author Response File: Author Response.docx

Reviewer 2 Report

Pedro et al. have reported diversity among isolates of Phytophthora infestans from potatoes using microsatellite markers. This topic is interesting, particularly given the ongoing evolution of pathogens and their hosts, which continually shapes the dynamic interactions between plants and pathogens. However, this manuscript lacks the critical data to substantiate the study's conclusions.

Below are comments and suggestions for improvement:

1. No images are provided for the plant study; the manuscript relies solely on data. The authors did not clarify which symptoms were used to collect infected leaves from potato fields, and no images were provided. Including these in the supplementary files would enhance the study's credibility. Additionally, the term "strategic points" is unclear and needs further explanation.
2. The authors mentioned that isolation was confirmed through macroscopic and microscopic observations, but they did not explain what these observations entailed or provide any image data. Since the isolates were collected from the wild, how was Phytophthora infestans confirmed? If identification was based solely on phenotype, particularly sporangia, it would have been better to perform ITS sequencing to confirm the strains.
3. The manuscript lacks a reference table for the disease scoring used to measure disease progression in inoculated plants. Furthermore, no image data is provided for the detachment assay or greenhouse studies.
4. No gel electrophoresis data is provided for the microsatellite markers used. Including these in the supplementary files would provide robust evidence to support the study's findings.
5. Please proofread the manuscript before submission. It appears that review comments from “Introduction”  were not deleted (see lines 22-30).
6. Formatting Issue: Ensure that the scientific names are italicised (e.g., line 10).

Pedro et al. have reported diversity among isolates of Phytophthora infestans from potatoes using microsatellite markers. This topic is interesting, particularly given the ongoing evolution of pathogens and their hosts, which continually shapes the dynamic interactions between plants and pathogens. However, this manuscript lacks the critical data to substantiate the study's conclusions.

Below are comments and suggestions for improvement:

1. No images are provided for the plant study; the manuscript relies solely on data. The authors did not clarify which symptoms were used to collect infected leaves from potato fields, and no images were provided. Including these in the supplementary files would enhance the study's credibility. Additionally, the term "strategic points" is unclear and needs further explanation.
2. The authors mentioned that isolation was confirmed through macroscopic and microscopic observations, but they did not explain what these observations entailed or provide any image data. Since the isolates were collected from the wild, how was Phytophthora infestans confirmed? If identification was based solely on phenotype, particularly sporangia, it would have been better to perform ITS sequencing to confirm the strains.
3. The manuscript lacks a reference table for the disease scoring used to measure disease progression in inoculated plants. Furthermore, no image data is provided for the detachment assay or greenhouse studies.
4. No gel electrophoresis data is provided for the microsatellite markers used. Including these in the supplementary files would provide robust evidence to support the study's findings.
5. Please proofread the manuscript before submission. It appears that review comments from “Introduction”  were not deleted (see lines 22-30).
6. Formatting Issue: Ensure that the scientific names are italicised (e.g., line 10).

Author Response

Please see the attachment

Author Response File: Author Response.docx

Reviewer 3 Report

General:

The manuscript describes the study of an interplay between the host resistance and pathogen virulence in the potato - Phytophthora infestans pair. Authors use a number of statistical methods to evaluate the obtained results. It is good, but one of the main questions for me is the substantiation of the purpose of the study (see below my comments for the Intro section) and the choice of isolates / collection points and cultivars used in the study as well as their number. Usually, such studies either include a large number of isolates and/or cultivars (that, in my opinion, would be a good choice in this case), or, if their number is limited, authors substantiate their choice. For example, they can use potato cultivars differing in their susceptibility to the studied disease, or the most common for the local production potato cultivars. However, the manuscript does not contain any information about this. Note that authors operate with such terms as the host resistance, but information about the level of this resistance is absent. At the same time, international standards suppose that each approved potato cultivar has been tested for its resistance to the late blight. Therefore, this information should be known for the cultivars included into the study, but authors did not provide it.

Concerning the number of isolates included into the study. One can use a limited number of isolates if the pathogen is represented in this location by only several clonal lineages (I suppose this is the case, but authors do not describe the situation in their country in such details, which would allow a reader to understand the choice of this small number of sampling sites and collected samples). However, in this case authors used only two differing isolates, though they really have 4 different pathogenicity patterns in relation to the infection development rate. Moreover, based on only two isolates, they conclude about a potential link between genetic distance and virulence and even about different defense mechanisms of potato plants in relation to such isolates. In my opinion, two points is a rather small number to make any conclusions about any correlations and relationships.

 I consider that the manuscript can not be published in the current form. In my opinion, authors should improve introduction section by adding information about similar studies and by clarifying the purpose of their study. This would allow us to conclude about the novelty of the obtained results. Authors should enlarge the range of isolates included into the study to add more points for correlation analysis and support of their conclusions and suggestions. They should add information about the late blight resistance level of chosen potato cultivars and substantiate their choice. Alternatively, they should explain why they used only two isolates as well as re-write the Discussion and Conclusion section to exclude some suggestions about correlations and relationships, which reliability and valuability in the case of use only two experimental points is doubtful.

There are also some comments concerning the description of materials and methods (see below): not all points are described quite clearly, so this section also requires some revision.

 Additional remark: Latin names of the pathogen and the host should be italic. Please, check the text and correct, if necessary (for example, line 10, 72, 87, 177, etc.).

TITLE

Too general title. I doubt authors mean the global study of all pathogen isolates and potato cultivars. I would recommend to modify it with allowance for the origin of the studied strains and (probably) cultivars).

ABSTRACT

It would be good to include information about the origin of isolates used in the study.

Line 14: “These isolates” - did you mean that only two isolates with the maximum genetic distance between them were used in the main part of the study, or you still tested 7 isolates?

INTRODUCTION

Lines 23-31: this text should be deleted.

Line 69-70: the statement on the objective of the study is poorly substantiated. Authors write that pathogenicity assays are essential for rapid identification and screening of resistant potato plants. At the same time, they did not give any information about the current situation in this sector of studies. Does anybody already worked on this problem? Which pathogenicity assays are already developed? Did anybody performed such studies for Colombian isolates and/or cultivars grown in this country? What is the need to study these seven isolates? Did authors wanted to evaluate pathogenicity of isolates from Colombia to get information about their potential harm? In this case, why did they took only seven isolates? Were these isolates important in any sense? Which potato cultivars were used in the study and why they were chosen? “Four different potato cultivars”: different in which features?

Therefore, I would strongly recommend authors to revise the manuscript, especially Intro section, to explain their choice of sampling sites, potato cultivars, and number of isolates and cultivars included into the study as well as to add information concerning the earlier performed studies in this field of science. Without this information it is impossible to evaluate the novelty and importance of results presented in this manuscript.

MATERIALS AND METHODS

The first arising question is: why authors spent three years to visit seven points in relatively small country to collect ONLY seven isolates??? Even if the local P. infestans populations are clonal and include only several clones, collection of a larger number of samples may potentially reveal any new genotype. I would expect to read rather that authors visited 7 points and collected 15-20-25 samples per a point; after isolation and primary identification, they revealed that only seven genotypes are present in the collected pool of samples, so the further investigation included 7 isolates representing all revealed genotypes.  Why did you choose these 7 points for sampling?

Line 88: how did you obtain mycelium from the agar plate? In the case when one culture P. infestans on liquid medium (pea medium, etc.), it is easy to obtain a piece of mycelium, which forms a mat on the surface of this medium (you just take this mat from the Petri plate, remove an agar piece (inoculum), wash mycelium with water to remove medium, and then take a piece. In the case of agar medium, you should use other techniques. Please, add information how you took a fragment of mycelium from the surface of agar plate.

Line 108-109: why did you mentioned this research group? Did you mean that the assay was carried out on their equipment or in their facilities or premises? Please, add information, since now this sentence looks slightly strange.

Line 113: which potato cultivar was used for this assay? Was it susceptible to P. infestans? How the inoculation occurred? Did you sprayed detached leaves with the suspension, or put small drops of it onto the leaf surface? Which surface, abaxial or adaxial, was used for inoculation. Please, add details.

Line 115: please, give more information about the calculation of the percentage of infection. Did you mean the area of lesions to the leaf area ratio or the percent of lesions developed from inoculated points, or something else?

Line 117: “… if there were differences between the treatments” - did you mean the difference between the treatments with different isolates?

Line 118: what is the symptom expansion rate and how did you calculate it? Please, remember that the description of methods should allow any user to reproduce your experiment, so it must include as many details as possible.

Line 126-129: why did you choose these potato cultivars for your experiment? Are they characterized by the late blight susceptibility, or represent a range of different susceptibility indices? What was the idea to use namely these 4 cultivars?

Line 131: four genotypes of potato? Or four genotypes of the pathogen? Please, specify.

Line 135: what is suspension without pathogen? Suspension of what? Please, explain. I suggest, the treatment of control plants could be performed by water.

Line 139: I suppose “plant genotype” would be more clear that “genotype”.

Line 141: why did you use only two pathogen isolates in this experiment? The results would be more interesting and reliable if you would include at least 3-4 isolates varied in their pathogenicity.

Line 148: please, give more detailed description of the method for measurement of the presence of necrotic spots in two different scales.

RESULTS

Line 189-191: authors could describe the similar studies (or declare the lack of such studies to accentuate the novelty of their own study) in the Introduction section. Right now the value of these results can not be evaluated. Do they completely new or even novel?

Line 192: since the term “genotype” can be referred to both pathogen and host, I would recommend authors to add the corresponding word to elucidate this issue.

Table 2: I suggest “Isolates” should be instead of “Isolated” (see Variation source column).

Line 196 and 197: there are two different explanations of the ”**” designation. Please, check and correct.

Line 213: are you sure that this difference is determined by different defense mechanisms? It can be determined by a simple difference in the number of resistance genes. Did you check this characteristic of the cultivars included into the study?

DISCUSSION

Line 232: actually, genetic distance between can be determined not only by changes in genes involved into the virulence of pathogens.

Line 233-237: please, substantiate this statement. For example, are there any publications about the association between any of the standard set of 12 SSR markers and effector genes related to the aggressiveness/pathogenicity/virulence of P. infestans?

Line 271-274: please, check the sentence as it seems to be incomplete.

CONCLUSION

In my opinion, such conclusions can not be made using only two different isolates of P. infestans. As I understand, the first pathogenicity assay for 7 isolates was made using only one (unknown0 potato cultivar. The second test, which included four different cultivars, was made only on two isolates.

Author Response

Please see the attachment

Author Response File: Author Response.docx

Round 2

Reviewer 1 Report

I went through both the revised manuscript from the authors and the cover letter that addressed all the comments and suggestions raised during the review process.    At this point,  I can boldly say that authors had addressed all what I pointed out and they equally effect the corrections within the manuscript.    Based on that,  I recommend the manuscript for acceptance as well as publication in IJPB. 

I recommend the manuscript for acceptance as well as publication in IJPB. 

Author Response

Dear Reviewer,

Thank you very much for your insightful comments and suggestions. They have significantly enriched the quality of the manuscript.

Reviewer 3 Report

Authors made a thorough work to answer the comments (or at least explain why they can not do this). It improved the manuscript. I still have some comments and hope authors will take it into account, especially the request for additional table (see comment to Table 2). It is not correct to give only statistical analysis without real data, since real data are the most valuable part of each paper, which allows reader to make comparisons with other results and improves the general understanding of the performed work. In relation to this table, authors should have the required data, so there is no need in any additional experiments.

 Comment:

 Potato variety or potato cultivar? Both terms are correct, but it would be good to use one of them across the whole text. Please, make your choice and correct.

Introduction

Line 62-63: please check the sentence. The most widely planted variety in Nariño is the most vulnerable of the four to attack by P. infestans The most widely planted cultivar - did you mean Capiro (which frequency is 38%)? “Of the four” of what? Cultivars? But you mentioned five potato cultivars. Did you mean “The most widely planted in Narino var. Capiro is the most susceptible to the late blight among the mentioned varieties…”?

Line 81: I suggest “resistant POTATO genotypes” would be better.

Results

Fig. 1A - it would be good to improve the distinguishability between the color designations of strains. Now it is not easy to distinguish, say, designations of P00519 and P00321, P00321 and P002521, P00119 and P02421 (thin lines and close colors). Possible solutions: (1) increase the thickness of lines or (2) make some lines from the similar group of colors to be dotted.

Table 2. 1) Since tables should be self-explanatory, it would be good to explain the “Df” designation. 2) Authors present only statistical treatment indices. It is incorrect. It is necessary to show initial data. Not mean squares, but real parameters estimated (number of affected leaflets, leaves, necrotic lesions, etc.). In this case, such table should be the basic, and the statistical table is just an auxilliary. In other case, a reader does not see real results, which are important for their comparison with any other studies. Please, add a table with the real estimated leaf parameters.

Line 247: … although he molecular bases… - I suppose it should be “the” instead of “he”.

Suppl. Fig. 16: it would be good to increase the brightness of pictures, because now they are too dark. 

Author Response

Please see the attachment.

Author Response File: Author Response.docx