Diversity of Phytophthora Species Detected in Disturbed and Undisturbed British Soils Using High-Throughput Sequencing Targeting ITS rRNA and COI mtDNA Regions

Round 1

Reviewer 1 Report

Overall comments: The paper refers to assesing Phytophthora diversity in British forests using high-throughput illumina sequencing targeting the widely accepted barcoding Internal Transcribed Spacer 1 (ITS1) region of rRNA and compared with the mitochondrial cytochrome c oxidase I (COI) gene. Also, isolation of Phytophthora was run in parallel.  The study was carried in disturbed sites DS (sites frequently visited by the public, with recent and new plantings or soil disturbances) and in undisturbed sites US (natural forest and woodland sites with little disturbance or management).  The results shows that Phytophthora species are present both in disturbed and undisturbed sites independent of the presence of declined trees, with a highest range of distribution into disturbed sites.

The novelty is reprezented by the first time work using COI to study Phytophthora community in soils. The results showed that COI primers were less specific than the ITS when targeting Phytophthora species. However, COI amplified more Oomycota and wider range of organisms than when using ITS primers. Also, the study showed the first detection of Nothophytophthora species in British soils in  both  disturbed and  undisturbed sites,  and  the  first  detection of  Nothophytophthora species using NGS analysis with both ITS and COI regions. Eight Phytophthora species were detected by both ITS and COI: P. cactorum,  P. cinnamomi, P. megasperma, P. plurivora, P. primulae, P. pseudosyringae, P. ramorum and P. syringae. The results indicate that although NGS analysis provides evidence of the presence of DNA of those Phytophthora spp., verification of those species, especially of those three new records for Britain should be attempted by re-sampling the sites and attempting to obtain living cultures from soil or from host material.

The paper is  well structured, the material and method section is wide and describes very well the methodology used. The discussions are in comparison with other researches and results. The references are adequate to the subject and recently. I find the conclusions are very clear and concise.

Also, very minor corrections are needed:

line 17… na-tural instead of nat-ural. Hyphenating in the middle of a syllable is regarded as a mistake.

line 43… a-gricultural or agri-cultural instead of ag-ricultural

line 353-358 … to use italic for all the Phytophthora species listed there.

Author Response

REVIEWER 1

Point 1: 1The paper is well structured, the material and method section is wide and describes very well the methodology used. The discussions are in comparison with other researches and results. The references are adequate to the subject and recently. I find the conclusions are very clear and concise.

Response 1:  Thank you for your comments.

Also, very minor corrections are needed:

Point 2: line 17… na-tural instead of nat-ural. Hyphenating in the middle of a syllable is regarded as a mistake.

Response 2: Thank you for pointing this out. The template of the journal cut the words automatically and we cannot control it. We hope that after the final editorial revision this will be solved. 

Point 3: line 43… a-gricultural or agri-cultural instead of ag-ricultural

Response 3: Thank you for pointing this out. The template of the journal cut the words automatically and we cannot control it. We hope that after the final editorial revision this will be solved.  

Point 4: line 353-358 … to use italic for all the Phytophthora species listed there.

Response 4: Thanks for noticing. We have corrected it

Additionally, we changed the order of Figures 2 and 3, since we made a mistake and they were interchanged.

Author Response File: Author Response.docx

Reviewer 2 Report

This interesting study is attempting to understand the baseline diversity of Phytophthora spp. in disturbed and natural soils within Great Britain.  Two different DNA markers, and culturing, were used to investigate the presence of the pathogen in soils collected from around the country.  The most important results from this work are not the descriptions of the diversity of Phytophthora spp., rather the issues that have been highlighted related to the methods and the results from the two DNA markers.  It is also interesting to note that neither method was 100% effective at detecting the species present in the control population.  The first item addressed in the Discussion is the difference between the two methods and the potential implications for studies based on one marker.  The Discussion also recommends that verification of these results should be confirmed by culturing for species that are new to Great Britain, specifically P. castaneae, P. capsici and P. fallax.   A strength of this manuscript is that the shortcomings of the methods are recognized, and in my opinion, this is the primary reason for my recommendation of acceptance of the manuscript.  If the intent of the manuscript was to provide an authoritative assessment of Phytophthora in Great Britain, without recognizing the shortcomings of the methods, my recommendation would not be as positive.  This is because analysis of the sample population of 10 species indicated that the methods were not 100% effective; therefore, one wonders what else has been missed?  At the minimum, the study indicates that Phytophthora are present in disturbed and undisturbed sites, and the populations between the two sites are different.  The lists that have been generated are also interesting as the species on the lists can be considered as present in Great Britain.  The finding that P. ramorum DNA could be detected post-eradication is also interesting and has regulatory implications.  For these reasons, my recommendation is that the manuscript be accepted following revision.

Items that require attention:

1. Abstract Line 33-34: “if these species are present”.  Although “confirm” is used twice in the previous sentence, suggest “There is a need to confirm the presence of these species and if they are a threat to British trees.”.  To “investigate if these species are present” suggests that the markers are giving false positive results.  Is there any evidence for false positives?
2. Table 1 is a mix of Materials and Methods, and Results. My recommendation is that results be removed from Table 1 and that the table only be used to describe the sites.  For the results, Supplementary Tables 1 and 2 are good.  Perhaps a summary table could be prepared for the body of the manuscript, without the number of reads and only the list of sites where each species was detected for the two markers combined.    
3. Line 163: “according to the according to the”
4. Lines 352 – 359: Add italics.
5. Line 356: Check spelling “ kermoviae”; suggest double checking all species are spelt correctly.
6. Lines 379 – 390 suggest that culturing can out-perform NGS, although this is not mentioned in the Discussion. There are important implications for this when designing experiments and storing samples for later analysis (i.e. viability of propagules should be considered) which should be noted in the Discussion section.
7. Are there other markers that should be used for Phytophthora that may have better efficacy? ATP9-NAD9?
8. Line 546 – 547: These are interesting results, what are the implications for regulators?
9. Line 548 “kalmia latifolia”; capitalize “K”.

Author Response

REVIEWER 2

Items that require attention:

Point 1: Abstract Line 33-34: “if these species are present”.  Although “confirm” is used twice in the previous sentence, suggest “There is a need to confirm the presence of these species and if they are a threat to British trees.”.  To “investigate if these species are present” suggests that the markers are giving false positive results.  Is there any evidence for false positives?

Response 1: That was true. We have rephrased it and clarify this statement since it was somehow redundant with the previous sentence. Now the sentence reads: “Additionally, there is a need to confirm if these species are a threat to British trees and try to establish any eradication measures required to mitigate Phytophthora spread in Britain.”

Point 2: Table 1 is a mix of Materials and Methods, and Results. My recommendation is that results be removed from Table 1 and that the table only be used to describe the sites.  For the results, Supplementary Tables 1 and 2 are good.  Perhaps a summary table could be prepared for the body of the manuscript, without the number of reads and only the list of sites where each species was detected for the two markers combined.    

Response 2: Thanks for the suggestion. We thought at the beginning to split Table 1 in two: One describing the sites and the second one with a summary of the results. However, we decided to combine both since otherwise this would duplicate the space needed for tables in the manuscript. We would like to leave the table as it is as it avoids readers searching for details on different tables.  

Point 3: Line 163: “according to the according to the”

Response 3: Corrected

Point 4: Lines 352 – 359: Add italics.

Response 4: Corrected

Point 5: Line 356: Check spelling “ kermoviae”; suggest double checking all species are spelt correctly.

Response 5: Corrected. We have also revised all species names and corrected all typos.

Point 6: Lines 379 – 390 suggest that culturing can out-perform NGS, although this is not mentioned in the Discussion. There are important implications for this when designing experiments and storing samples for later analysis (i.e. viability of propagules should be considered) which should be noted in the Discussion section.

Response 6: We believe that this paragraph does not try to describe that culturing can out-perform NGS. Nevertheless, we have included a sentence to clarify this point on lines 492-494 of the Discussion: Special care should be taken for processing samples, since soil storage could decrease viability of propagules and might diminish the success of pathogen isolation.

Point 7: Are there other markers that should be used for Phytophthora that may have better efficacy? ATP9-NAD9?

Response 7: We have added a sentence concerning this on lines 476—478: It would be interesting to test whether other genes that have shown good for discrimination of Phytophthora spp. isolates when using pure cultures [32,38], may be used in NGS analysis and provide complementary results to that obtained for COI and ITS.

Point 8: Line 546 – 547: These are interesting results, what are the implications for regulators?

Response 8: We have added a sentence in relation to this point on lines 556-558: These results indicate that re-sampling the sites where some Phytophthora outbreaks have occurred would be advisable to test whether viable propagules of the pathogen are still present on those soils.    

Point 9: Line 548 “kalmia latifolia”; capitalize “K”.

Response 9: Corrected

Additionally, we changed the order of Figures 2 and 3, since we made a mistake and they were interchanged.

Author Response File: Author Response.docx