Diversity and Distribution of Phytophthora Species in Protected Natural Areas in Sicily
, Antonella Pane 3, Francesco Aloi 3,4, Maria Evoli 3, Marilia Horta Jung 1,2, Bruno Scanu 5, Roberto Faedda 3, Cinzia Rizza 3, Ivana Puglisi 3, Gaetano Magnano di San Lio 6, Leonardo Schena 6 and Santa Olga Cacciola 3,*Round 1
Reviewer 1 Report
In this manuscript, Jung and colleagues investigate diversity and distribution of Phytophthora species within protected natural areas in Sicily. For that purpose, they combined morphological studies of Phytophthora isolates with ITS sequencing. The results presented here constitute an important dataset for pest management. However, other than the dataset itself I do not see any scientific novelty reported here.
Please note that high compression levels make all figures hard to read!
Minor edition:
line 314: please remove the underscore/underline before 'were'
Author Response
Manuscript forests-457140
Diversity and distribution of Phytophthora species in Protected Natural Areas in Sicily
RESPONSES TO EDITORS AND REVIEWERS
We are very grateful to Editors and Reviewers for their precious suggestions and criticisms which greatly contribute to improve the manuscript. In this document we list in detail the answer to all comments and suggestions. Please note that the revision introduced by the authors are in red color in the manuscript as well as reported here in quotes after each comment of the reviewers.
Comment of Reviewer 1
Comment
In this manuscript, Jung and colleagues investigate diversity and distribution of Phytophthora species within protected natural areas in Sicily. For that purpose, they combined morphological studies of Phytophthora isolates with ITS sequencing. The results presented here constitute an important dataset for pest management. However, other than the dataset itself I do not see any scientific novelty reported here.
Response
In our opinion, the results presented in this study made it possible for us as well as for the scientific community to have the first overall picture on the complexity of Phytophthora species in Sicily. The study provides not only a detailed dataset but also improves our knowledge about the attitude of Phytophthora populations to live in peculiar natural ecosystems (i.e. plant communities characterized by a specific geographic location and geological substrate). Furthermore, the sampling activities carried out in the framework of this study have allowed the first isolation of Phytophthora vulcanica, a recently described cryptic species reported only from volcano Etna, and the first isolation in Sicily of Phytophthora tyrrhenica, previously recovered only in Sardinia. These two new species were described also by some of the authors of this manuscript in Jung et al., IMA Fungus 2017 and cited in the revised manuscript.
Comment
Please note that high compression levels make all figures hard to read!
Response
We agree with the referee and although high resolution images have been already provided as separated files during the submission of the manuscript, it seems they were lost. So, the original high resolution images will be submitted again.
Comment
Line 314: please remove the underscore/underline before 'were'
Response
It was done (line 316 of the revised manuscript)
Author Response File: 
Author Response.pdf
Reviewer 2 Report
This manuscript describes the occurrence of Phytophthora species across a set of ecologicaly interesting sites in the mediterranean island of Sicily. The results are quite clear and will be ion interest to those in closely related fields. I have no major concern but suggest some minor changes:
(1) Line 33 and elsewhere, I don't think you need capital letters for 'internal transcribed spacer'.
(2) Line 36. What exactly are these "new insights"? The paper generates lots of observations but the insights seems to be lost in there. It would be helpful to the reader to clearly and succinctly state the insights.
(3) Line 65. Is it really exponential? Or is it linear but with a very steep slope?
(4) Line 71. "occurrence and ecology ... have" not "has".
(5) Most of the images are of poor quality in the PDF for review. I hope these will be improved in the final version.
(6) In the Results section the authors often refer to the clades of Phytophthora. How are these defined? Are the clades based on ITS? In that case, how can it be that on line 401 a species is assigned to a clade based on 'multigene phylogenetic study'? Please explictly explain the Phytophthora clade system and cite appropriate references.
(7) Line 163. Similarity? Or identity?
(8) Line 164. Cite reference for kelmania?
(9)Line 285. The authors distinguish their study from previous ones by saying that those previous were metabarcoding approaches. I don't think this is the important distinction. The important distinction is that the present study involved culturing whilst the previous studies did not culture the taxa.
(10)Line 357. Is this group really expanding? Or is it simply our knowledge and awareness of the group that is expanding?
Author Response
Manuscript forests-457140
Diversity and distribution of Phytophthora species in Protected Natural Areas in Sicily
RESPONSES TO EDITORS AND REVIEWERS
We are very grateful to Editors and Reviewers for their precious suggestions and criticisms which greatly contribute to improve the manuscript. In this document we list in detail the answer to all comments and suggestions. Please note that the revision introduced by the authors are in red color in the manuscript as well as reported here in quotes after each comment of the reviewers.
Comment of Reviewer 2
Comment
(1) Line 33 and elsewhere, I don't think you need capital letters for 'internal transcribed spacer' (see lines 33 and 149).
Response
It was done (see lines 32 and 150 of the revised manuscript)
Comment
(2) Line 36. What exactly are these "new insights"? The paper generates lots of observations but the insights seems to be lost in there. It would be helpful to the reader to clearly and succinctly state the insights
Response
We used the term "insight" as synonym of "knowledge". The paper provides numerous new knowledge about the distribution, host associations and ecology of Phytophthora species. Therefore, we modified the text as follows (see lines 35-36 of the revised manuscript): “New knowledge about the distribution, host associations and ecology of several Phytophthora species was provided”.
Comment
(3) Line 65. Is it really exponential? Or is it linear but with a very steep slope?
Response
The increase in the number of described Phytophthora species over time has been represented by a mathematical function in a Cartesian coordinate system (n. of described species vs time) in the paper of "Kroon, L.P.N.M.; Brouwer, H.; de Cock, A.W.A.M.; Govers, F. The Genus Phytophthora Anno 2012. Phytopathology 2012, 102, 348–364." (see figure 1). Although the authors of the aforementioned paper don't define the kind of represented mathematical function, it is not linear and its trend appears very similar to that of an exponential function. Anyway, the term was changed in “dramatically” (see line 65 of the revised manuscript).
Comment
(4) Line 71. "occurrence and ecology ... have" not "has".
Response
It was done (see line 71 of the revised manuscript)
Comment
(5) Most of the images are of poor quality in the PDF for review. I hope these will be improved in the final version.
Response
We agree with the referee, we report here the same answer given to reviewer 1: <although high resolution images have been already provided as separated files during the submission of the manuscript, it seems they were lost. So, the original high resolution images will be submitted again>.
Comment
(6) In the Results section the authors often refer to the clades of Phytophthora. How are these defined? Are the clades based on ITS? In that case, how can it be that on line 401 a species is assigned to a clade based on 'multigene phylogenetic study'? Please explicitly explain the Phytophthora clade system and cite appropriate references.
Response
We agree with the observation of the referee; in fact, we used the phylogenetic nomenclature of clades based on the multigenic approach of Jung et al., 2018. Therefore, the division of the genus Phytophthora into well-supported clades results not only from ITS sequencing, but on the sequence of several loci as also shown in Blair et al., 2008, Fungal Genetics and Biology 45: 266–277. Therefore, we highlighted this aspect in the introduction (see lines 48-50 of the revised manuscript): “With more than 150 described species grouped in twelve multigenic phylogenetic Clades [6], this oomycete genus comprises some of the most aggressive plant pathogens of forests and other natural ecosystems [7-16]”.
Comment
(7) Line 163. Similarity? Or identity?
Response
We thank the reviewer for this suggestion and agree with his observation, therefore we used the more appropriate term identity in the text (see line 165 of the revised manuscript).
Comment
(8) Line 164. Cite reference for kelmania?
Response
It was done (see line 167 of the revised manuscript)
Comment
(9) Line 285. The authors distinguish their study from previous ones by saying that those previous were metabarcoding approaches. I don't think this is the important distinction. The important distinction is that the present study involved culturing whilst the previous studies did not culture the taxa.
Response
We agree with the observation of the referee; in fact, in the original manuscript we reported as follow (lines 284 - 287): ‘This is the first study of Phytophthora diversity in Europe using conventional isolation methods and covering both a wide range of natural forest types and water courses crossing these areas. Previously, the only surveys of Phytophthora diversity in both forests and rivers within the same region in Europe used a metabarcoding approach which is based on a DNA identification technologies and high-throughput DNA sequencing’ (in red color, the modified text of the revised manuscript: lines 288-289).
Comment
(10) Line 357. Is this group really expanding? Or is it simply our knowledge and awareness of the group that is expanding?
Response
We agree with the observation of the referee. Therefore, we modified the text as follows (see lines 355-357 of the revised manuscript): ‘The number of known species of Clade 2 is rapidly increasing; besides P. plurivora and P. multivora it includes numerous other aggressive Phytophthora species’.
Author Response File: Author Response.pdf
