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I was not able to obtain a 2nd review from the reviewers who was more critical of the manuscript in the first submission. Therefore I made some additional comments for clarification and corrected a number of typos in your re-submission. Please consider my suggestions and edits in the attached document, and resubmit a final version that I will accept.
Grammar and copy editing has been improved significantly.
Additions of several references have been made which improve reporting and clarity.
Experimental design is now much clearer.
Methods for strain selection and identification are clear and repeatable. Inclusion of raw data in SI is very helpful.
The authors responded very well to my concerns, and I think the manuscript is much improved by these (and other) changes. I thank the authors for fully clarifying the methods and experimental design, including great supporting info for the Fusarium strain(s), and for improving figure/table captions.
The reviewers make many valid comments and suggestions. Importantly, you must better justify why one or two isolates of each species, collected from different hosts or locations, can be used to study local adaptation. More clarity and transparency is required in your methods and results. Please ensure that you are following PeerJ policies regarding supporting data. In addition, your resubmission will have to be more carefully edited. I noted many cases of sentences missing periods in the introduction (see attached pdf).
Generally the article is written clearly. There are some typos and unclear sentences, which have been highlighted in the attached pdf. There are several places where a digit is used when it is more appropriate for the number to be spelled out fully (four, ten, etc.). Figure 1 uses red, green, and blue to present information, which is not ideal as it makes it likely color-blind people will not be able to read the figure and the figure will be unclear when printed in greyscale. I recommend changing to colors with differing saturation instead of hue as that solves both problems.
The authors aimed to investigate the factors that could contribute to variation in pathogen resistance in tomatoes and using a generalized linear model found that several climatic factors combined best predicted pathogen resistance.
I found this generally to be an informative and interesting paper on factors contributing to tomato pathogen resistance. Sections of the discussion could be made clearer, but otherwise well written as well.
The manuscript is formatted well and generally readable, but there are minor but pervasive grammar issues that sometimes add confusion to, or at least distraction from, the main story. It would likely benefit from editing by a native English speaker.
Introduction and background seem thorough and relevant for the scope of the study.
Some clarity could be added to certain figure/table captions (described in specific comments).
Raw data concerning pathogen infection rates does not appear to be available, though location metadata is in SI. Making raw data about response variable available would ensure that statistical results could be validated. Additionally, sequence data from the pathogen strains used are not available.
Pathogen screening method is generally appropriate and justified. There should be more clarity about pathogen strain selection, in particular the Fusarium sp. strain. It is not clear from the methods or the linked website, what isolation steps were taken to ensure axenic culture of Fusarium sp. or how spore densities were determined. Further, B-Tubulin sequence data for the three strains needs to be made archived and referenced in the manuscript along with the parameters for determining taxonomy from sequence data and morphology. There are thousands of strains called "Fusarium sp," for example, and providing seq data (including methods) and morphological methods (maybe micrographs in SI?) for your pathogen strains is essential.
There could also be more clarity regarding the physical setup of the experimental units (even just some photos in SI).
Statistical methods are generally appropriate for the hypotheses being tested and are well-documented. I do like to see proper citations for R packages that form major components of project analyses so the developers of those tools/methods get proper attribution, so please consider citing statistical packages.
Findings are appropriate given the results, and are well-tied to background context. Very thoughtful use of caveats to conclusions and speculation is restrained and clearly denoted as such.
This is a generally well-done study and I think the results are interesting. It does need some work on clarity, methods reporting, and raw data availability before it is ready to be published, in my opinion.
Some specific comments follow:
some grammar and editing issues, e.g., line 62 "generates therefore" -> "therefore generates";
line 66: missing "."
line 77: "thus" ??
line 85: variationS
line 122: "top10"
line 162: "with off the agar"
These (and others) do not sink the manuscript, but need to be corrected before publication. Thorough copy editing and grammar check from native English speaker would be helpful.
Line 162: please define "mesh" including pore size, etc.
Lines 173-178: I would like to know more about how they determined the genus of their Fusarium isolate. Is the Beta Tubulin gene sequence deposited in an appropriate repository? Fusarium species (and strains) have a very wide range of pathogenicity and host specificity. Proper identification of the strain used in this study is crucial. Also, the authors state that these isolates were re-cultivated fro several rounds on PDA for cleanup, but do not mention how they determined that the cultures were axenic. This is important to ensure that the Fusarium sp. was ONLY Fusarium sp.
The link to lab protocols listed on Line 180 did not clear this up
Lines 185-188: I am a bit confused about the physical layout of the experiment. Perhaps some supplemental figure(s) showing layout of the boxes would help.
Line 200: R version? Also, it is nice to directly cite R packages that were crucial to analyses, rather than just mentioning that they are in CRAN. This also helps clarify package versions, etc.
Line 201: Proper citation for R package is... Douglas Bates, Martin Maechler, Ben Bolker, Steve Walker (2015). Fitting Linear Mixed-Effects Models Using lme4. Journal
of Statistical Software, 67(1), 1-48. doi:10.18637/jss.v067.i01.
Line 210: Proper citation for R package is... Torsten Hothorn, Frank Bretz and Peter Westfall (2008). Simultaneous Inference in General Parametric Models. Biometrical
Journal 50(3), 346--363.
ggplot2 citation: H. Wickham. ggplot2: Elegant Graphics for Data Analysis. Springer-Verlag New York, 2009.
gplots: Gregory R. Warnes, Ben Bolker, Lodewijk Bonebakker, Robert Gentleman, Wolfgang Huber Andy Liaw, Thomas Lumley, Martin
Maechler, Arni Magnusson, Steffen Moeller, Marc Schwartz and Bill Venables (2016). gplots: Various R Programming Tools for
Plotting Data. R package version 3.0.1. https://CRAN.R-project.org/package=gplots
Line 216: please cite maps package (I don't have it installed, but it can be found in R with "citation("maps")")
Line 237-238: Not totally clear as to what "effects" of surface sterilization they are talking about. Cannot find figure description for Fig S1 to describe what I am seeing.
Line 276: "a varient of" ... please be more specific
Line 300: Need to define AIC before first use; also, need to explain how AIC values were derived
Lines 305-310: This may be confusingly worded, or I may just misunderstand. If "genetic group" is a better indicator of infection rates, I don't see an obvious link to conclusions about "micro-environments." It would seem that genetic groupings are not independent of climate variables in this case.
Line 317: same issue as above
Line 318: The information in S data 2 is a bit "unruly" and difficult to parse. Readers would benefit from clearer labeling. For instance, the models are still denoted by the variable names used in R and it is not readily clear which model goes with which conclusion from the main manuscript. Perhaps the P-values should be reported directly in the manuscript instead of the SI.
Lines 338-345: This conclusion seems to contradict the results stated on line 306, but that section was confusing to me. Perhaps another well-designed figure could go a long way to clarifying the relationships between genetic group, location, and pathogen susceptibility.
Lines 372-380: I appreciate the very good caveats about climate data
Table 1: It is not clear why "model 1" and "model 6" are in bold?
Overall the manuscript has several punctuations, formatting, and grammatical errors. Some scientific names are not in italics, while words that should not be in italics are italicized.
Line 82. The authors claim that that in this study they ‘aim to investigate the occurrence of spatially heterogeneous evolutionary pressures between populations of Solanum chilense, a solanaceous wild species, and several pathogens in a relatively small geographical space which exhibits large variation in habitat quality and abiotic environmental factors.’ However, one or two strains per pathogen species seems to be a small number to access evolutionary pressures between plants and pathogens.
Line 183. The authors state that the leaves were of the same age. Are the plants also of the same age?
Line 194: Were the four repetitions done on different days? That is, where the biological repetitions done with the same set of plants. Or was it done with plants that were independently grown at a different time?
Line: 197 What do the authors mean with ‘dependent on temperature and growth conditions’? Where these changed throughout the experiment?
Line 226: ‘We selected seven populations that represent three previously described genotype groups.’ This paragraph should be in the ‘methods’ section.
Line 235: How did the authors measure the surface coating of the leaves?
Line 237: Are there references that show that washing leaves with 70% ethanol eliminates the surface coating? Why were the authors interested in eliminating the surface coating if this might make a difference in the resistance of the distinct cultivars or populations of S. chilense? Might surface coating be an adaptation to differences in environmental conditions between populations of S. chilense? What were the effects of ‘surface sterilization’? Less contamination or better infection efficiency? Was this properly assessed or was it merely qualitative?
Line 242: The authors seem to be focused only on vertical resistance. Why are they not considering horizontal resistance as well? The fact that the authors talk about resistance against multiple pathogens makes horizontal resistance, more plausible. Also, if vertical resistance is being investigated, why are they just assessing two strains of Alternaria solani, and one strain of Phytophthora infestans and of Fusarium? This is not representative of the pathogen’s population.
Based on Figure 2, the authors mention that there are differences in ‘infection rates’ among populations. However, it is not clear which populations are significantly different.
It the authors are assessing vertical resistance; climatic variables should not affect the performance of the plants. It seems that authors might also be considering horizontal resistance where environment might play a role in differential gene expression. Thus, plants grown at different times should have been assessed. Also, ideally, plants should have been grown in the field given that they are expected to be primed as opposed to plants grown under green house conditions.
It is not clear how climatic variables were selected? Also, it is difficult to associate results from this study with what is being discussed. Despite the fact that the populations of S. chilense are originally found in regions with distinct climatic variables, the plants used in this study were all grown under the same controlled environmental conditions. Thus, conclusions such as “our results show indeed that temperature in winter as well as temperature in the wettest quarter have a significant effect on infection rate”. This was not explicitly assessed in this study.
Authors conclude in line 397: “In this study, several mechanisms theoretically proposed to generate stable long term polymorphism at host resistance and pathogen infectivity loci are shown to originate from the environmental heterogeneity across populations.” It is not clear to me how the evidence presented supports this conclusion.
I wonder if the conclusions are valid when the authors are using a strain of Alternaria solani from Germany. Does this reflect the coevolution between the populations of S. chilense assessed and the pathogen population they are exposed to?
I do not believe it is appropriate to make conclusions based on a single isolate. Also, have this isolates been conserved on media for several years? If so, it is important to ‘activate’ them by transferring them through a susceptible host prior to the experimental inoculations.
It is not clear whether you happened to find Fusarium in the green house by chance? If so, then this individual was not collected from the region where the plants are originally from.
The aim of this study was to show specific pathogen adaptations between populations of S. chilense through inoculation assays using three plant pathogens: Alternaria solani, Phytophthora infestans, and Fusarium sp.). The authors seem to focus on vertical resistance but the evidence presented seems to support horizontal resistance.
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