Six First Reports of Pin Nematodes from Portugal, with an Update of the Systematics, Genetic Diversity, and Phylogeny of the Genus Paratylenchus (Nematoda: Tylenchulidae)
, Maria L. Inácio 2, Jonathan D. Eisenback 3 and Carlos Gutiérrez-Gutiérrez 1,*Round 1
Reviewer 1 Report
A very detailed exhaustive manuscript on the morphometric and molecular identification of six Paratylenchus species. I congratulate the authors for their approach especially for using three distinct genes for a comparative study. Such an integrative approach to species discrimination deserves appreciation. I have made some minor corrections on the ms itself which may be taken care of.
Comments for author File: 
Comments.pdf
Author Response
Reviewer 1:
Response to Reviewer 1:
Thank you for the meticulous and detailed reading of our manuscript “Six first reports of pin nematodes from Portugal, with an update of the systematics, genetic diversity, and phylogeny of the genus Paratylenchus (Nematoda: Tylenchulidae)”.
We thank the Reviewer for these positive comments. We went through the manuscript and addressed most of the reviewer’s suggestions.
Thank you once again for your advice and review.
Point 1. Page 2/ line 63. To delete “moderately”.
Response 1: Corrected
Point 2. Page 2/ line 63. To delete “and unambiguous”.
Response 2. Corrected.
Point 3. Page 3/ line 127. To delete “of Paratylenchidae”.
Response 3. Corrected.
Point 4. Page 3/ line 137. Pin nematode species It should not be Pin species.
Response 4. Corrected and checked all text.
Point 5. Page 3/ line 143. Underlined “from four major grapevine-growing areas of Central and South Portugal”.
Response 5. It is explained in detail in Material and Method (page 4/line 156: four of the main grapevine-growing regions (Alentejo, Tejo, Setubal and Lisbon)).
Point 6. Page 3/ line 147. Pin nematode species It should not be Pin species:
Response 6. Corrected.
Point 7. Page 5/ line 183. “It is better to use the term diameter rather than width because the nematodes are round. Now a days in taxonomic description of soil nematodes, the term diameter is more commonly used”.
Response 7. “Width” is replaced by “diameter”. Corrected.
Point 8. Page 9/line 312. To replace “three four juvenile-stage” by “three 4th stage juvenile”.
Response 8. Corrected.
Point 9. Page 9/line 313. If males not found, how the authors write males had a few differences.
Response 9. “Males” is replaced by “Juveniles”. Corrected.
Point 10. Page 10/line 321. Pin nematode species It should not be Pin species.
Response 10. Corrected.
Point 11. Page 10/line 354. To replace “width” by “wide.
Response 11. Corrected and checked all text.
Point 12. Page 13/line 388. Pin nematode species It should not be Pin species.
Response 12. Corrected.
Point 13. Page 16/line 460. Pin nematode species It should not be Pin species: Corrected.
Response 13. Corrected.
Point 14. Page 29/line 822. Pin nematode species It should not be Pin species: Corrected.
Response 14. Corrected.
Point 15. Page 29/line 826. Text underlined “This comparative morphological taxonomic study of the eight Portuguese populations of Paratylenchus spp. confirmed that the identification of pin nematode species from phenotypic features is not easy …”. Although the authors mention that the identification of Pin nematode species based on phenotypic features is not easy but in this paper all the six species were identified based on the morphometric identification key Ghaderi et al 2014.
Response 15. It is a mistake. “This comparative morphological taxonomic of the eight Portuguese populations” is replaced by “This integrative taxonomic study of eight Portuguese populations”. Corrected.
Reviewer 2 Report
Horticulturae – 1683274
Six first reports of pin nematodes from Portugal, with an update of the systematics, genetic diversity, and phylogeny of the genus Paratylenchus (Nematoda: Paratylenchidae)
The manuscript describes the results of nematode survey in vineyards in Central an Southern Portugal, focused on nematodes of the genus Paratylenchus.
The manuscript brings a comprehensive review on Paratylechus, systematics, diversity, biology, host range, world distribution and agricultural importance.
Overall, the study brings important contribution to the field of plant nematology, since for the first time the occurrence of six species do Paratylenchus is revealed for Portugal, all of them associated with grape plants. Species identification was unequivocally performed by simultaneous morphological, morphometric and molecular approaches, and supported by phylogenetic evidence. In addition, new reliable molecular markers were developed.
The text, tables and figures are clear and properly presented. Although, minor repairs are needed according to the comment below, as pointed for line/row numbers.
Page 1
Line 29 – I suggest removing the authority for Vitis vinifera L., since species authorities were not considered for the nematode species names.
Linee 31/32 – For the Keywords, I recommend to remove the words Paratylenchus spp. and pin nematodes, since they appear in the title.
Line 35 – I suggest inserting the authority after Tylenchida (first time placed in the text)
Line 38 – I recommend inserting the authority after Paratylenchus
Lines 35-41 – The authors did not consider using De Ley & Blaxter`s nematode classification system, which has been widely accepted (De Ley, P.; Blaxter, M.L. A new system for Nematoda: Combining morphological characters with molecular trees and translating clades into ranks and taxa. Nematol. Monogr. Perspect. 2004, 2, 633–653;
Ahmed, M.; Holovachov, O. Twenty Years after De Ley and Blaxter—How Far Did We Progress in Understanding the Phylogeny of the Phylum Nematoda? Animals 2021, 11, 3479. https://doi.org/10.3390/ ani11123479)
Page 2
Line 54 – please, use the correct symbol for `Celsius degree`
Line 63 – the word `moderately` is misplaced – I suggest removing it
Line 78 – `pin nematode species` seems more appropriate
Lines 84 and 86 – please insert que year of publication after Goodey, and Oostenbrink,
Page 3
Line 54 – please, use the correct symbol for `Celsius degree`
Line 63 – the word `moderately` is misplaced – I suggest removing it
Line 114 and 137 – `pin nematode species` seems more appropriate
Page 5
Line 182– the sentence ` lip region and width` seems to be incomplete (lip region height and width???)
Lines 215-216 – please replace `ul` by the correct unit (µl)
Page 6
Lines 271-272– please, insert the authority, year of publication after P. variabilis (first time in the text)
Line 272 – please remove the letter `s` after P. veruculatus
Page 9
Line 306-307(Figure 1) – please insert parentheses (arrowhead) and correct `arrowed` to arrowhead
Line 312 – please remove the word `four`
Line 313 – please replace `detected.. Males….` by: detected. Juveniles….
Page 10
Line 321 – `pin nematode species` seems more appropriate
Page 13
Lines 380-381 (Figure 2) – please replace `arrowed` by arrowhead
Lines 388-382 – please correct to: … (Vitis vinifera)
Line 395 – please insert the authority, year of publication after P. baldacii (first time in the text)
Page 14
Line 411 (Figure 3)- Figures 3E, 3M and 3N are not Light micrographs, they are Scanning Electron Microscopy micrographs
Lines 412-415 (Figure 3) – please replace `arrowed` by arrowhead
Page 15
Line 435 (Table 4)- please correct `(Vitis vinifera L) to: (Vitis vinifera L.)
Page 17
Lines 468 – please correct `Raski 1975` to: Raski, 1975
Page 18
Line 516 – please change `belong` to: belonging
Lines 527 (Figure 4) – please replace `arrowed` by arrowhead
Page 19
Lines 551-552 (Figure 5) – please replace `arrowed` by arrowhead.
Page 20
Lines 561-563 – the text is confused, mainly for the reader who is not familiar to Portugal geography. It says: This Portuguese population was found in one soil sample of grapevine in commercial vineyards with unknown rootstock in São Domingos de Carmões locality, Torres Vedras municipality, and Lisbon district (Southern Portugal) (Table 1). I understand that `one soil sample` belongs to a single locality, but in the way the text was written there are two localities related to the same soil sample: 1. Vedras municipality, and 2. Lisbon district (which is not in Table 1). Therefore, I recommend an improved wording.
Line 570 – please add the authority, year of publication after P. recisus (first time in the text)
Line 578 – please correct `records` to: record
Page 21
Lines 607-608 (Figure 6) – please replace `arrowed` by arrowhead
Lines 613-616 – the text is also confused. It says: This Portuguese population was found in one soil sample of grapevine (Vitis vinifera L.) in a commercial vineyard with unknown rootstock in Monte da Ribeira, São Manços, and Évora district in the Southern Portugal (Table 1). I understand that `one soil sample` belongs to a single locality, but in the way the text was written there are two localities related to the same soil sample: 1. Monte da Ribeira, São Manços, and 2. Évora district (which is not in Table 1). Therefore, I recommend an improved wording.
Page 22
Line 621 – please change `to these` to: from these
Lines 629-630 – please correct `records` to: record
Line 631 – please change `on the Iberian Peninsula` to: in the Iberian Peninsula
Line 638 – please use italic typing for Paratylenchus
Line 639 – please correct `P. variabiliti to: P. variabilis
Page 23
Line 699 – please correct `agree` to: agrees
Page 24
Line 732 – please correct to: Hemicycliophora
Page 25
Line 789 – please remove the word `were`
Page 28
Figure 9 – somewhere in the text it should be mentioned that Paratylenchus tenuicaudatus (Portugal population) was not included in phylogenetic analysis inferred from COI sequences.
Page 29
Line 816 – there is no need of table citation in Discussion text.
Page 30
Lines 867, 877, 882, 889, 891, 892, 895 – please change (um) to the correct unit (μm)
Lines 869, 870, 883 – please add the authorities, year of publication after: P. leptus, P. rostrocaudatus, P. aquaticus, P. enigmaticus (first time in the text).
Line 881 – please correct `a several species` to: several species
Line 885 – please correct `agree with` to: agrees with..
Line 897 – please correct `to found a same… to: to find the same…
Line 902 – please correct `multiples evolutionary synapomorphies`: multiple evolutionary synapomorphies
Line 905 – please correct `to feed of` to: to feed in
Line 907 – please correct `do not need` to: does not need
Lines 908-910 – suggestion of replacement text: P. veruculatus has a short and rigid stylet that allows this nematode to feed in root cells of outside layers in thin lateral roots of herbaceous and woody plants and need to move drilling new feeding sites.
Page 31
Line 914 – suggestion of replacement text: Paratylenchus species infesting grapevine soils in Portugal.
Lines 937-938 – suggestion of replacement text: All authors have read and agreed to publish this version of the manuscript.
Lines 943-944 – suggestion of replacement text: …and I. Ferreira from the Instituto Mediterrâneo para a Agricultura, Ambiente e Desenvolvimento (MED), Universidade of Évora for his excellent technical assistance.
Author Response
Reviewer 2:
Response to Reviewer 2:
Thank you for the meticulous and detailed reading of our manuscript “Six first reports of pin nematodes from Portugal, with an update of the systematics, genetic diversity, and phylogeny of the genus Paratylenchus (Nematoda: Tylenchulidae)”.
We thank the Reviewer for these positive comments. We went through the manuscript and addressed most of the reviewer’s suggestions.
Thank you once again for your advice and review.
Point 0. Page 1/ Line 29 – I suggest removing the authority for Vitis vinifera L., since species authorities were not considered for the nematode species names.
Response 0: “Vitis vinifera L.” is replaced by “grapevine”
Point 1. Page 1/ Line 31-32 – For the Keywords, I recommend to remove the words Paratylenchus spp. and pin nematodes, since they appear in the title.
Response 1: “Paratylenchus spp. and pin nematodes” are replaced by morphology and taxonomy. Corrected.
Point 2. Page 1/ Line 35 – I suggest inserting the authority after Tylenchida (first time placed in the text)
Response 2: Corrected.
Point 3. Page 1/ Line 38 – I recommend inserting the authority after Paratylenchus
Response 3: Corrected.
Point 4. Page 1/ Lines 35-41 – The authors did not consider using De Ley & Blaxter`s nematode classification system, which has been widely accepted (De Ley, P.; Blaxter, M.L. A new system for Nematoda: Combining morphological characters with molecular trees and translating clades into ranks and taxa. Nematol. Monogr. Perspect. 2004, 2, 633–653; Ahmed, M.; Holovachov, O. Twenty Years after De Ley and Blaxter—How Far Did We Progress in Understanding the Phylogeny of the Phylum Nematoda? Animals 2021, 11, 3479. https://doi.org/10.3390/ ani11123479)
Response 4: I have incorporated this information in the text. Corrected.
Point 5. Page 2/ Line 54 – please, use the correct symbol for `Celsius degree`
Response 5: “ºC” is replaced by “°C”. Corrected.
Point 6. Page 2/ Line 63 – the word `moderately` is misplaced – I suggest removing it
Response 6: I have been removed it. Corrected.
Point 7. Page 2/ Line 78 – `pin nematode species` seems more appropriate
Response 7: Corrected and checked all text.
Point 8. Page 2/ Lines 84 and 86 – please insert que year of publication after Goodey, and Oostenbrink,
Response 8: “P. peraticus (Raski, 1962) Siddiqi & Goodey, 1964,….., P. straeleni (de Coninck, 1931) Oostenbrink, 1960,….”. Corrected.
Point 9. Page 3/Line 54 – please, use the correct symbol for `Celsius degree`
Response 9: I think that it was corrected in the point 4. I didn´t found it in the page 3.
Point 10. Page 3/Line 63 – the word `moderately` is misplaced – I suggest removing it
Response 10: I think that it was corrected in the point 5. I didn´t found it in the page 3.
Point 11. Page 3/Line 114 and 137 – `pin nematode species` seems more appropriate
Response 11: Corrected and checked all text.
Point 12. Page 5/ Line 182 – the sentence ` lip region and width` seems to be incomplete (lip region height and width???)
Response 12: “tail length and width” is replaced by “tail length and diameter”. Corrected.
Point 13. Page 5/Lines 215-216 – please replace `ul` by the correct unit (µl)
Response 13: Corrected and checked all text.
Point 14. Page 6/ Lines 271-272– please, insert the authority, year of publication after P. variabilis (first time in the text)
Response 14: “P. variabilis Raski, 1975 …”. Authority and year of publication inserted in the text. Corrected.
Point 15. Page 6/ Line 272 – please remove the letter `s` after P. veruculatus
Response 15: Corrected.
Point 16. Page 9/ Line 306-307(Figure 1) – please insert parentheses (arrowhead) and correct `arrowed` to arrowhead
Response 16: Corrected and checked all text.
Point 17. Page 9/ Line 312 – please remove the word `four`
Response 17: “four juvenile-stage (J4)…” is replaced by “4th stage juvenile (J4) …”. Corrected.
Point 17. Page 9/ Line 313 – please replace `detected.. Males….` by: detected. Juveniles….
Response 18: “Males…” is replaced by “Juveniles…”. Corrected.
Point 19. Page 10/ Line 321 – `pin nematode species` seems more appropriate
Response 19: Corrected and checked all text.
Point 20. Page 13/ Lines 380-381 (Figure 2) – please replace `arrowed` by arrowhead
Response 20: Corrected and checked all text
Point 21. Page 13/ Lines 388-382 – please correct to: … (Vitis vinifera)
Response 21: Corrected.
Point 22. Page 13/ Line 395 – please insert the authority, year of publication after P. baldacii (first time in the text)
Response 22: P. baldaccii Raski, 1975,… was cited first time in the text in page 2 line 80. However, the name was incorrect. “P. baldacii” is replaced by “P. baldaccii”. Corrected and checked in all text.
Point 23. Page 14/Line 411 (Figure 3) Figures 3E, 3M and 3N are not Light micrographs, they are Scanning Electron Microscopy micrographs
Response 23: Figure 3. Light micrographs of Paratylenchus pedrami ..” is replaced by“Figure 3. Light and scanning electron microscopy micrographs of Paratylenchus pedrami….”. Corrected.
Point 24. Page 14/Lines 412-415 (Figure 3) – please replace `arrowed` by arrowhead
Response 24: Corrected and checked in all text.
Point 25. Page 15/ Line 435 (Table 4)- please correct `(Vitis vinifera L) to: (Vitis vinifera L.)
Response 25: Corrected and checked in all text.
Point 26. Page 17/ Lines 468 – please correct `Raski 1975` to: Raski, 1975
Response 26: Corrected.
Point 27. Page 18/ Line 516 – please change `belong` to: belonging
Response 27: Corrected.
Point 28. Page 18/ Lines 527 (Figure 4) – please replace `arrowed` by arrowhead
Response 28: Corrected and checked in all text.
Point 29. Page 19/ Lines 551-552 (Figure 5) – please replace `arrowed` by arrowhead.
Response 29: Corrected and checked in all text.
Point 30. Page 20/ Lines 561-563 – the text is confused, mainly for the reader who is not familiar to Portugal geography. It says: This Portuguese population was found in one soil sample of grapevine in commercial vineyards with unknown rootstock in São Domingos de Carmões locality, Torres Vedras municipality, and Lisbon district (Southern Portugal) (Table 1). I understand that `one soil sample` belongs to a single locality, but in the way the text was written there are two localities related to the same soil sample: 1. Vedras municipality, and 2. Lisbon district (which is not in Table 1). Therefore, I recommend an improved wording.
Response 30: This Portuguese population was found in one soil sample of grapevine in commercial vineyards with unknown rootstock in São Domingos de Carmões locality, Torres Vedras (Lisbon district, Southern Portugal). Corrected.
Point 31. Page 20/ Line 570 – please add the authority, year of publication after P. recisus (first time in the text)
Response 31: “P. recisus Siddiqi, 1996 …”. Corrected.
Point 32. Page 20/ Line 578 – please correct `records` to: record
Response 32: Corrected.
Point 33. Page 21/ Lines 607-608 (Figure 6) – please replace `arrowed` by arrowhead
Response 33: Corrected.
Point 34. Page 21/ Lines 613-616 – the text is also confused. It says: This Portuguese population was found in one soil sample of grapevine (Vitis vinifera L.) in a commercial vineyard with unknown rootstock in Monte da Ribeira, São Manços, and Évora district in the Southern Portugal (Table 1). I understand that `one soil sample` belongs to a single locality, but in the way the text was written there are two localities related to the same soil sample: 1. Monte da Ribeira, São Manços, and 2. Évora district (which is not in Table 1). Therefore, I recommend an improved wording.
Response 34: This Portuguese population was found in one soil sample of grapevine (Vitis vinifera L.) in a commercial vineyard with unknown rootstock in Monte da Ribeira locality, São Manços (Évora district, Southern Portugal). Corrected.
Point 35. Page 22/Line 621 – please change `to these` to: from these
Response 35: Corrected.
Point 36. Page 22/Lines 629-630 – please correct `records` to: record
Response 36: Corrected.
Point 37. Page 22/Line 631 – please change `on the Iberian Peninsula` to: in the Iberian Peninsula
Response 37: Corrected.
Point 38. Page 22/Line 638 – please use italic typing for Paratylenchus
Response 38: Corrected.
Point 39. Page 22/Line 639 – please correct `P. variabiliti to: P. variabilis
Response 39: Corrected.
Point 40. Page 23/ Line 699 – please correct `agree` to: agrees
Response 40: Corrected.
Point 41. Page 24/ Line 732 – please correct to: Hemicycliophora
Response 41: Corrected.
Point 42. Page 25/Line 789 – please remove the word `were`
Response 42: Corrected.
Point 43. Page 28/ Figure 9 – somewhere in the text it should be mentioned that Paratylenchus tenuicaudatus (Portugal population) in phylogenetic analysis inferred from COI sequences.
Response 43: This information has been included in the text (see page 24/line751). A total of eleven new sequences that include all the Portuguese populations of Paratylenchus spp. except for P. tenuicaudatus were obtained for this mitochondrial ribosomal molecular marker and included in this phylogenetic analysis. Corrected.
Point 44. Page 29/ Line 816 – there is no need of table citation in Discussion text.
Response 44: Table citation has been deleted. Corrected.
Point 45. Page 30/ Lines 867, 877, 882, 889, 891, 892, 895 – please change (um) to the correct unit (μm)
Response 45: Corrected.
Point 46. Page 30/ Lines 869, 870, 883 – please add the authorities, year of publication after: P. leptus, P. rostrocaudatus, P. aquaticus, P. enigmaticus (first time in the text).
Response 46: Corrected in the text.
Point 47. Page 30/ Line 881 – please correct ` with a several species` to: several species
Response 47: “…with a several species…` is replaced by “to several species”. Corrected.
Point 48. Page 30/ Line 885 – please correct `agree with` to: agrees with..
Response 48: Corrected in the text.
Point 49. Page 30/ Line 897 – please correct `to found a same… to: to find the same…
Response 49: Corrected in the text.
Point 50. Page 30/ Line 902 – please correct `multiples evolutionary synapomorphies`: multiple evolutionary synapomorphies
Response 50: Corrected in the text.
Point 51. Page 30/ Line 905 – please correct `to feed of` to: to feed in
Response 51: Corrected in the text.
Point 52. Page 30/ Line 907 – please correct `do not need` to: does not need
Response 52: Corrected in the text.
Point 53. Page 30/ Lines 908-910 – suggestion of replacement text: P. veruculatus has a short and rigid stylet that allows this nematode to feed in root cells of outside layers in thin lateral roots of herbaceous and woody plants and need to move drilling new feeding sites.
Response 53: I agree with your suggestion. Corrected in the text.
Point 54. Page 31/ Line 914 – suggestion of replacement text: Paratylenchus species infesting grapevine soils in Portugal.
Response 54: I agree with your suggestion. Corrected in the text.
Point 55. Page 31/ Lines 937-938 – suggestion of replacement text: All authors have read and agreed to publish this version of the manuscript.
Response 55: I agree with your suggestion. Corrected in the text.
Point 56. Page 31/ Lines 943-944 – suggestion of replacement text: …and I. Ferreira from the Instituto Mediterrâneo para a Agricultura, Ambiente e Desenvolvimento (MED), Universidade of Évora for his excellent technical assistance.
Response 56: I agree with your suggestion. Corrected in the text.
