ORIGINAL RESEARCH published: 03 October 2018 doi: 10.3389/fmicb.2018.02334
Differential Usefulness of Nine Commonly Used Genetic Markers for Identifying Phytophthora Species Xiao Yang* and Chuanxue Hong Hampton Roads Agricultural Research and Extension Center, Virginia Tech, Virginia Beach, VA, United States
Edited by: Hector Mora Montes, Universidad de Guanajuato, Mexico Reviewed by: Henrik R. Nilsson, University of Gothenburg, Sweden Bernardo Franco, Universidad de Guanajuato, Mexico *Correspondence: Xiao Yang
[email protected] Specialty section: This article was submitted to Fungi and Their Interactions, a section of the journal Frontiers in Microbiology Received: 03 August 2018 Accepted: 12 September 2018 Published: 03 October 2018 Citation: Yang X and Hong C (2018) Differential Usefulness of Nine Commonly Used Genetic Markers for Identifying Phytophthora Species. Front. Microbiol. 9:2334. doi: 10.3389/fmicb.2018.02334
The genus Phytophthora is agriculturally and ecologically important. As the number of Phytophthora species continues to grow, identifying isolates in this genus has become increasingly challenging even by DNA sequencing. This study evaluated nine commonly used genetic markers against 154 formally described and 17 provisionally named Phytophthora species. These genetic markers were the cytochrome-c oxidase 1 (cox1), internal transcribed spacer region (ITS), 60S ribosomal protein L10, beta-tubulin (β-tub), elongation factor 1 alpha, enolase, heat shock protein 90, 28S ribosomal DNA, and tigA gene fusion protein (tigA). As indicated by species distance, cox1 had the highest genus-wide resolution, followed by ITS, tigA, and β-tub. Resolution of these four markers also varied with (sub)clade. β-tub alone could readily identify all species in clade 1, cox1 for clade 2, and tigA for clades 7 and 8. Two or more genetic markers were required to identify species in other clades. For PCR consistency, ITS (99% PCR success rate) and β-tub (96%) were easier to amplify than cox1 (75%) and tigA (71%). Accordingly, it is recommended to take a two-step approach: classifying unknown Phytophthora isolates to clade by ITS sequences, as this marker is easy to amplify and its signature sequences are readily available, then identifying to species by one or more of the most informative markers for the respective (sub)clade. Keywords: oomycetes, plant disease diagnosis, plant pathology, genetics, plant destroyers
INTRODUCTION The genus Phytophthora currently consists of approximately 200 formal and provisional species with many high-impact plant pathogens (Erwin and Ribeiro, 1996; Yang et al., 2017). For example, P. infestans and P. sojae are major threats to potato and soybean production, respectively (Erwin and Ribeiro, 1996). Phytophthora ramorum (Goheen et al., 2002; Rizzo et al., 2002, 2005) and P. cinnamomi (Zentmyer, 1980; Shearer et al., 2004) are destructive forest pathogens causing tree decline in the U.S. and Australia, respectively. Identifying Phytophthora isolates to species is the first and critical step to support plant biosecurity. This process is now done primarily by DNA sequencing. Concerted efforts have been made to identify genetic markers and improve the accuracy of DNA sequence-based identification. As a result, a variety of markers have been identified and utilized (Cooke et al., 2000; Martin and Tooley, 2003; Kroon et al., 2004; Blair et al., 2008; Robideau et al., 2014). Meanwhile, many signature sequences from ex-types (type-derived cultures) and authentic isolates (representative isolates designated by the originators of the respective species) have been generated (Cooke et al., 2000; Kroon et al., 2004; Blair et al., 2008; Martin et al., 2014; Yang et al., 2017), although their
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Yang and Hong
Evaluating Markers for Identifying Phytophthoras
The mass was blotted dry on sterile tissue paper, transferred to a R -24 (MP Biomedicals, garnet bead tube and lysed in a FastPrep R 16 Santa Ana, CA). gDNA was purified using a custom Maxwell R FFS nucleic acid extraction kit in combination with a Maxwell Rapid Sample Concentrator (Promega, Madison, WI). A pair of primers including the forward primer ITS6 and reverse primer ITS4 (Cooke et al., 2000) was used to amplify the ITS region. The cox1 fragment was amplified with the primer pair COXF4N and COXR4N (Kroon et al., 2004). PCR reaction mixtures were prepared with Takara Taq DNA polymerase (Takara Shuzo, Shiga, Japan) according to the manufacturer’s instructions. Each cox1 PCR reaction mixture contained an additional 2-µL 25 mM MgCl2 and 0.25-µL Bovine serum albumin (BSA) per 25-µL. Thermal cycling protocols were described previously (Cooke et al., 2000; Kroon et al., 2004). All PCR products were evaluated for successful amplification using agarose gel electrophoresis. Sequencing reactions were run in both directions with the same primer pairs used for amplification at the University of Kentucky Advanced Genetic Technologies Center (Lexington, KY) or Eton Bioscience Inc. (Durham, NC). Results were viewed in Finch TV version 1.4.0 (Geospiza, Seattle, WA), aligned using Clustal X (Larkin et al., 2007), and edited manually to correct obvious sequencing errors and code ambiguous sites according to the International Union of Pure and Applied Chemistry (IUPAC) nucleotide ambiguity codes to produce a consensus sequence. All sequences produced in this study have been deposited in GenBank (Table 1). Rates of PCR success for all nine genetic markers were estimated by calculating the percentage of successful amplifications over all PCR reactions performed by the authors for each marker during the past 6 years.
availability in public repositories depends upon species (Kang et al., 2010). These two lines of advancement have raised several questions of practical importance. What genetic markers are most useful? Is their resolution dependent upon (sub)clade? How many markers are required to identify Phytophthora isolates within a respective (sub)clade to species? Answers to the above and other related questions will help identifying Phytophthora species accurately in the timeliest fashion and at the lowest cost. To this end, Martin et al. (2012) indicated that a set of genetic markers may be required for the most accurate identification. These included the internal transcribed spacer region (ITS), 60S ribosomal protein L10 (60S), beta-tubulin (β-tub), elongation factor 1 alpha (EF-1α), enolase (ENL), heat shock protein 90 (Hsp90), 28S ribosomal DNA (28S), tigA gene fusion protein (tigA), cytochrome-c oxidase 1 and 2 (cox1 and cox2), subunit 9 of NADH dehydrogenase (nad9), ribosomal protein S10 (rps10), and SecY protein (secY) coding regions. Correspondingly, reference sequences from various markers have been compiled for many known Phytophthora species (Cooke et al., 2000; Kroon et al., 2004; Blair et al., 2008; Grünwald et al., 2011; Park et al., 2013; Martin et al., 2014; Yang et al., 2017). In separate studies, Martin et al. (2014) and Martin and Tooley (2003) provided the average pairwise species distances for the concatenated nuclear and mitochondrial genes, and five mitochondrial markers, namely cox1&2, nad9, rps10, and secY. The objectives of this study were to evaluate nine commonly used genetic markers against more than 170 Phytophthora taxa and identify the most informative markers for individual (sub)clades.
MATERIALS AND METHODS Genus-Wide Distance Analyses
Sequence Selection
All nine genetic markers were analyzed for overall species distances resolved across the genus Phytophthora. Sequence datasets of each marker were aligned using the MUSCLE version 3.7 (Edgar, 2004) in MEGA version 7.0.26 (Kumar et al., 2016). Alignments were manually modified when obvious errors were present. The alignment of each marker was then trimmed to an equal size and question marks were inserted to represent missing data at both ends of short sequences. DNA sequence distances were calculated using the Kimura 2-parameter (K2P) distance model (Kimura, 1980) to explore the maximum, minimum and mean distances across the genus.
Nine common genetic markers, namely ITS, cox1, 60S, β-tub, EF-1α, ENL, Hsp90, 28S, and tigA, were evaluated. Sequences of 180 Phytophthora isolates representing 154 described and 17 provisionally named species were analyzed. These included 116 ex-types and 28 authentic isolates (Table 1). Eight taxa were represented by two or three isolates due to the lack of sequence data for all regions of individual isolates. The majority of 60S, β-tub, EF-1α, ENL, Hsp90, 28S, and tigA sequences originated from two previous studies (Blair et al., 2008; Yang et al., 2017). ITS and cox1 sequences of 90 and 79 Phytophthora species, respectively, were downloaded from GenBank (Benson et al., 2018). Sequences from P. sp. ohioensis (ST18-37) were obtained from the Phytophthora Database (Park et al., 2013). Seventy-nine and 86 isolates were sequenced for ITS and cox1, respectively in this study as described below to fill the signature sequence gaps in current public repositories.
Distance Analyses Within Individual (Sub)Clades Four selected markers that had relatively high mean species distances across the genus (cox1, ITS, tigA, and β-tub) were analyzed for distances within individual (sub)clades. Phylogenetic (sub)clade assignments for each species were identified according to the recent study by Yang et al. (2017). Sequence datasets within individual (sub)clades of each marker were aligned and edited as described above. Maximum, minimum, and mean distances within individual (sub)clades of each marker were calculated as described above.
DNA Extraction, Amplification, and Sequencing To extract genomic DNA (gDNA), a 5 × 5 mm agar plug was cut from the actively growing edge of a fresh culture and transferred to 20% clarified V8 broth. Cultures were incubated at room temperature (c. 23◦ C) for 7–14 d to produce a mycelial mass.
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3
30G8
P. tentaculata
2b
554.88
122779
62B4
42B2
45G4
41B7
P. glovera
P. mengei
P. mexicana
P. siskiyouensis
MYA-4187
MYA-4554 46731
121969
22F4
P. capsici
15399, MYA-4034
92550 P15122
P0646
P11685
133865
65B8
P. terminalis
P. amaranthi
101557
65B9
P. occultans
T
T
T
A
T
T
T
Nepal
Hawaii, USA
Virginia, USA
Taiwan
The Netherlands
The Netherlands
Brazil
1995
1948
2008
2010
1998
1987
Stream water
Solanum lycopersicum
Oregon, USA
Argentina
2003
n.a.
Persea americana California, USA n.a.
Nicotiana tabacum
Capsicum annum New Mexico, USA
Amaranthus tricolor
Pachysandra terminalis
Buxus sempervirens
2005
2005
2000
1966
North Carolina, n.a. USA
Hevea brasiliensis India
219.88
61J9
P. meadii
129185
Quercus leucotricophora
128767
61G2
P. himalsilva
T
Colocasia esculenta
n.a.
1999
1992
2001
2004
1969
1985
1975
1987
2001
n.a.e
Year
Delaware, USA 2000
Mexico
Hevea brasiliensis Malaysia
35D3
T
P. colocasiae
P3425 Irrigation water
581.69
62C6
Nicotiana tabacum
03E5
15410, MYA-4037
P. botryosa
2a
22F9
P. citrophthora
P. nicotianae
Phaseolus lunatus
Mirabilis jalapa
23B4
A
P. phaseoli
P3006
30C1
Mexico
Ecuador
Germany
Iran
P. mirabilis
Solanum brevifolium
Argyranthemum frutescens
Solanum melongena
Australia
Oregon, USA
Ipomoea Mexico longipedunculata
T
A
T
T
Trifolium subterraneum
Psendotsuga menziesii
31B5
P10225
P13365
P3882
T
T
Scotland, UK
P. ipomoeae
136915
158964
287317
P3943
P10339
Rubus idaeus
The Netherlands
Ohio, USA
Location
Solanum tuberosum
64069, MYA-4062
MYA-3655
60237
58713, 60438 278933
331662
T A
Viburnum sp.
Rhododendron sp.
Host or substrate
27A8
109229
374.72
347.86
52938
313728 313727
T
Typec
P. infestans
P. andina
P. andina
61J4
32G1
P. iranica
P. clandestina
P. clandestina
1
1c
1b
P. pseudotsugae
MYA-4065
P6767
971.95
34D4
P. idaei
P. idaei
P19523
111725
62A5
P. hedraiandra
WPC P10194
IMI
22E6
ATCC
P. cactorum
CBS
1a
CH
Species
(Sub) cladea
Isolate identificationb
TABLE 1 | Information and GenBank accession numbers of isolates used in this study.
KF317102
MH620024
MH620023
MH620022
KF317094
n.a.
JX978168
MH620021
AY564192
MH620020
KF317097
KF317096
MH620019
KF317091
MH620018
MH620017
MH620016
KC733447
AY564160
MH620015
AY564189
AY564172
AY564199
AY564185
AY769115
MH620014
cox1
KF317081
MH620111
EU748545
MH620110
KF317073
GU111585
JX978167
JX978155
MH620109
MH620108
KF317076
KF317075
MH620107
KF317070
MH620106
MH620105
MH620104
KC733443
FJ801734
MH620103
MH620102
MH620101
FJ802112
FJ801946
AY707987
MH620100
ITS
KX250677
KX250670
KX250656
KX250649
KX250635
n.a.
KX250607
KX250600
KX250593
KX250572
KX250565
KX250544
KX250537
KX250509
KX250495
KX250481
EU080830
KX250474
EU080182
KX250453
KX250439
EU079866
EU080426
EU080129
KX250397
KX250369
60S
KX250678
KX250671
KX250657
KX250650
KX250636
KJ179949
KX250608
KX250601
KX250594
KX250573
KX250566
KX250545
KX250538
KX250510
KX250496
KX250482
EU080831
KX250475
EU080183
KX250454
KX250440
EU079867
EU080427
EU080130
KX250398
KX250370
β-tub
KX250679
KX250672
KX250658
KX250651
KX250637
n.a.
KX250609
KX250602
KX250595
KX250574
KX250567
KX250546
KX250539
KX250511
KX250497
KX250483
EU080832
KX250476
EU080184
KX250455
KX250441
EU079868
EU080428
EU080131
KX250399
KX250371
EF-1α
KX250680
KX250673
KX250659
KX250652
KX250638
n.a.
KX250610
KX250603
KX250596
KX250575
KX250568
KX250547
KX250540
KX250512
KX250498
KX250484
EU080833
KX250477
EU080185
KX250456
KX250442
EU079869
EU080429
EU080132
KX250400
KX250372
ENL
GenBank accession no.d
KX250681
KX250674
KX250660
KX250653
KX250639
n.a.
KX250611
KX250604
KX250597
KX250576
KX250569
KX250548
KX250541
KX250513
KX250499
KX250485
EU080834
KX250478
EU080186
KX250457
KX250443
EU079870
EU080430
EU080133
KX250401
KX250373
Hsp90
KX250682
KX250675
KX250661
KX250654
KX250640
n.a.
KX250612
KX250605
KX250598
KX250577
KX250570
KX250549
KX250542
KX250514
KX250500
KX250486
EU080835
KX250479
EU080187
KX250458
KX250444
EU079871
EU080431
EU080134
KX250402
KX250374
28S
(Continued)
KX250683
KX250676
KX250662
KX250655
KX250641
n.a.
KX250613
KX250606
KX250599
KX250578
KX250571
KX250550
KX250543
KX250515
KX250501
KX250487
EU080836
KX250480
EU080188
KX250459
KX250445
EU079872
EU080432
EU080135
KX250403
KX250375
tigA
Yang and Hong Evaluating Markers for Identifying Phytophthoras
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4
4
3
2e
2d
2c
(Sub) cladea
MYA-4084
T
Quercus robur
784.95
30A5
P. quercina
Theobroma cacao Soil
MYA-4038
Theobroma cacao
15C7
T
Soil
Soil
P. quercetorum
202077
Western Australia, Australia
Germany
Germany
Oregon, USA
n.a.
n.a.
2012
2009
Germany
1995
South Carolina, 1997 USA
Costa Rica
Cameroon
Western Australia, Australia
Western Australia, Australia
n.a.
1995
1997
2008
KF358241
KF358240
KF317108
MH620035
KJ396688
MH620034
KF317106
KF358239
KF317105
MH620033
KF317104
California, USA n.a.
KF317103
MH620032
KF317098
MH620031
JX524159
2006
n.a.
2001
2004
MH620030
MH620029
KF317101
KF317100
MH620028
FJ237508
KF317095
MH620027
MH620026
n.a.
MH620025
cox1
n.a.
The Netherlands
Virginia, USA
The Netherlands
Eucalyptus dunnii South Africa
Quercus robur
Quercus robur
Rainwater
Lithocarpus densiflorus
Ilex aquifolium
Stream water
Cymbidium sp.
2005
1998
1925
2008
2007
1987
n.a.
2010
1969
n.a.
Year
North Carolina, 1999 USA
South Africa
Western Australia, Australia
Minnesota, USA
UK
Western Australia, Australia
Taiwan
South Africa
Italy
Brazil
Hawaii, USA
Location
Eucalyptus smithi South Africa
Soil
Fragaria × ananassa
Agathosma betulina
Kalmia latifolia
Pinus resinosa
Acuba japonica
Soil
Citrus sinensis
Curtisia dentata
Acer pseudoplatanus
Theobroma cacao
Macadamia integrifolia
22G9
42100
T
A
T
T
T
T
T
A
T
T
T
T
A
A
T
T
T
T
T
T
A
T
P. palmivora
238.83
P16948
P10410
P10117
P0716
P1819
P0630
WPC
Host or substrate
61J5
138637
502404
21173
IMI
Typec
P. megakarya
P. boodjera
127950
55C2
P. arenaria
803.95
29J5
P. psychrophila
121939
MYA-4222
30A8
P. pseudosyringae
47G5
MYA-4930
111772
60B3
P. pluvialis
P. alticola
MYA-2948
41C4
MYA-4577
P. nemorosa
114348
62A7
38J5
P. taxon aquatilis
545.96
P. ilicis
29E3
47G8
P. multivesiculata
P. frigida
125799
61F2
P. taxon emzansi
55C4
22E9
P. plurivora
P. elongata
45F1
P. pini
122081
61H7
P. pachypleura
31E6
MYA-3657
124094
55C5
P. multivora
P. bisheria
64532
128319
221.88
62C1
33H8
P. capensis 60440
46705
76651, MYA-4218
ATCC
P. citricola
133931
434.91
35C8
61H1
CBS
CH
Isolate identificationb
P. acerina
P. sp. brasiliensis
P. tropicalis
Species
TABLE 1 | Continued
KF358229
KF358228
KF317086
MH620121
KJ372244
MH620120
KF317084
KF358227
KF317083
MH620119
KF317082
JX524158
FJ666126
MH620118
KF317077
MH620117
MH620116
MH620115
KF317080
KF317079
MH620114
FJ237521
KF317074
MH620113
JX951285
GU259388
MH620112
ITS
KX252654
KX251062
KX251055
KX251034
n.a.
KX251013
KX251006
KX250992
KX250978
KX250971
KX250964
KX250950
KX250929
EU080065
KX250915
KX250894
EU080741
KX250859
KX250817
KX250810
KX250789
KX250775
KX250747
KX250726
KX250712
EU080419
KX250698
60S
KX252655
KX251063
KX251056
KX251035
KJ372283
KX251014
KX251007
KX250993
KX250979
KX250972
KX250965
KX250951
KX250930
EU080066
KX250916
KX250895
EU080742
KX250860
KX250818
KX250811
KX250790
KX250776
KX250748
KX250727
KX250713
EU080420
KX250699
β-tub
KX252656
KX251064
KX251057
KX251036
n.a.
KX251015
KX251008
KX250994
KX250980
KX250973
KX250966
KX250952
KX250931
EU080067
KX250917
KX250896
EU080743
KX250861
KX250819
KX250812
KX250791
KX250777
KX250749
KX250728
KX250714
EU080421
KX250700
EF-1α
KX252657
KX251065
KX251058
KX251037
KJ396738
KX251016
KX251009
KX250995
KX250981
KX250974
KX250967
KX250953
KX250932
EU080068
KX250918
KX250897
EU080744
KX250862
KX250820
KX250813
KX250792
KX250778
KX250750
KX250729
KX250715
EU080422
KX250701
ENL
GenBank accession no.d
KX252658
KX251066
KX251059
KX251038
KJ396710
KX251017
KX251010
KX250996
KX250982
KX250975
KX250968
KX250954
KX250933
EU080069
KX250919
KX250898
EU080745
KX250863
KX250821
KX250814
KX250793
KX250779
KX250751
KX250730
KX250716
EU080423
KX250702
Hsp90
KX252659
KX251067
KX251060
KX251039
n.a.
KX251018
KX251011
KX250997
KX250983
KX250976
KX250969
KX250955
KX250934
EU080070
KX250920
KX250899
EU080746
KX250864
KX250822
KX250815
KX250794
KX250780
KX250752
KX250731
KX250717
EU080424
KX250703
28S
(Continued)
KX252660
KX251068
KX251061
KX251040
n.a.
KX251019
KX251012
KX250998
KX250984
KX250977
KX250970
KX250956
KX250935
EU080071
KX250921
KX250900
EU080747
KX250865
KX250823
KX250816
KX250795
KX250781
KX250753
KX250732
KX250718
EU080425
KX250704
tigA
Yang and Hong Evaluating Markers for Identifying Phytophthoras
October 2018 | Volume 9 | Article 2334
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6b
6a
5
(Sub) cladea
5 MYA-4456
131653
132023
61G8
60B2
P. bilorbang
P. borealis
140357
129424
127951
554.67
66D1
55B6
62B8
34A8
P. crassamura
P. fluvialis
P. gibbosa
P. gonapodyides
P. chlamydospora
131652
61G6
P. amnicola
60351
P6872
T
T
T
T T
MYA-4881 28765
T
T
A
P16851
T A A
389736
P11555
P. taxon walnut
40A7
47J1
P. sp. personii
P. sp. personii
P. rosacearum
Western Australia, Australia
Western Australia, Australia
Spain
Taiwan
The Netherlands
Western Australia, Australia
Western Australia, Australia
Western Australia, Australia
Malaysia
Hawaii, USA
Taiwan
New Zealand
Ohio, USA
Location
2013
2010
1996
1976
1999
2008
2011
2011
n.a.
1990
n.a.
2006
2006
Year
Reservoir water
Soil
Stream water
Soil
Soil
Prunus sp.
Stream water
Soil
Stream water
Irrigation water
Nicotiana tabacum
2008
2010
2009
2006
n.a.
Western Australia, Australia
Western Australia, Australia
Italy
Western Australia, Australia
1967
2009
KC733448
MH620046
MH620044 MH620045
2012
HQ012878
MH620043
MH620042
MH620041
MH620040
MF326861
MH620039
MF326858
MF326847
KF112863
KF112862
MH620038
HQ012881
MF326843
MF326863
AY564182
MH620037
AY564190
MH620036
n.a.
cox1
2009
1995
United Kingdom 1971
Alaska, USA
Western Australia, Australia
Western Australia, Australia
Virginia, USA
North Carolina, n.a. USA
Malus domestica California, USA n.a.
Soil
143061
P. pseudorosacearum
Olea sp. Soil
T
Soil
Zostera marina
143060
390121
T
A
P. kwongonina
30J3
200.81
32F8
P. humicola
P. inundata
123382
Soil
143062
P. cooljarloo
46H1
Soil
143059
P. condilina
P. gemini
Soil
143058
Heavae sp.
Cocos nucifera
Soil
Agathis australis
Soil
Host or substrate
P. balyanboodja
P3826
T
52179, MYA-4080
T 180616
22J1
296.29
67D6
T T
P. heveae
P15175
ST18-37 A
WPC
P. cocois
325914
IMI
67D5 36818
ATCC
61J7
587.85
CBS
Typec
P. agathidicida
CH
Isolate identificationb
P. castaneae
P. sp. ohioensis
Species
TABLE 1 | Continued
60S β-tub
EF-1α
ENL
Hsp90
28S tigA
KF112854
MH620130
MH620129
KP863493
AF541890
MH620128
MH620127
MH620126
MH620125
FJ801604
MH620124
KJ372267
JN547636
KF112856
KF112855
FJ217680
HQ012957
KJ372262
KJ372258
MH620123
KP295304
MH620122
KP295308
KX251236
KX251222
KX251208
KX251201
JF273125
KX251187
KX251181
KX251167
KX251452
EU080312
KX251445
n.a.
n.a.
KX251153
KX251139
KX251125
n.a.
n.a.
n.a.
KX251111
KX251104
KX251097
KX251076
KX251237
KX251223
KX251209
KX251202
KF750602
KX251188
KX251182
KX251168
KX251453
EU080313
KX251446
MF326827
MF326824
KX251154
KX251140
KX251126
MF326816
MF326814
MF326806
KX251112
KX251105
KX251098
KX251077
KX251238
KX251224
KX251210
KX251203
n.a.
KX251189
n.a.
KX251169
KX251454
EU080314
KX251447
n.a.
n.a.
KX251155
KX251141
KX251127
n.a.
n.a.
n.a.
KX251113
KX251106
KX251099
KX251078
KX251239
KX251225
KX251211
KX251204
n.a.
KX251190
KX251183
KX251170
KX251455
EU080315
KX251448
n.a.
n.a.
KX251156
KX251142
KX251128
n.a.
n.a.
n.a.
KX251114
KX251107
KX251100
KX251079
KX251240
KX251226
KX251212
KX251205
HQ012922
KX251191
KX251184
KX251171
KX251456
EU080316
KX251449
MF326878
MF326876
KX251157
KX251143
KX251129
HQ012925
MF326869
MF326892
KX251115
KX251108
KX251101
KX251080
KX251241
KX251227
KX251213
KX251206
GU594846
KX251192
KX251185
KX251172
KX251457
EU080317
KX251450
n.a.
n.a.
KX251158
KX251144
KX251130
n.a.
n.a.
n.a.
KX251116
KX251109
KX251102
KX251081
(Continued)
KX251242
KX251228
KX251214
KX251207
n.a.
KX251193
KX251186
KX251173
KX251458
EU080318
KX251451
n.a.
n.a.
KX251159
KX251145
KX251131
n.a.
n.a.
n.a.
KX251117
KX251110
KX251103
KX251082
Phytophthora Phytophthora Phytophthora Phytophthora Phytophthora Phytophthora Phytophthora Phytophthora Database Database Database Database Database Database Database Database
ITS
GenBank accession no.d
Yang and Hong Evaluating Markers for Identifying Phytophthoras
October 2018 | Volume 9 | Article 2334
Frontiers in Microbiology | www.frontiersin.org
6
7b
7a
6
(Sub) cladea
43F3
P. × stagnum
52854
45F3
31E7
60A4
P. melonis
P. niederhauserii
P. pisi
582.69
44388
45F7
133347
61H3
P. cajani
46719, MYA-4076
P. asiatica
P. × multiformis
141207
141209
67C1
67C2
P. × heterohybrida
22F6
P. × cambivora
P. × incrassata
47A7
P. × alni
109054
392316
392314
P10617
P3105
P16202
P10413
T
A
T
T
T
T
T
T
T
62A3
P. uliginosa
P. uniformis
T T
67B9
46C7
P. intricata
P. rubi
T P6231
T
209.46
67C4
61J3
P. formosa
P. fragariae
T T
109049
62A2
67C3
P. europaea
T
A
T
T
T
T
T
T
T
T
T
T
T
P. flexuosa
P6306
P16100
P3599
P10337
WPC
141199
181417
32035
389725
IMI
67C5
90442
MYA-4826
MYA-4926
MYA-4882
MYA-4946
58817
ATCC
Typec
P. attenuata
P. sp. sulawesiensis
132095
127954
55C1
P. thermophila
62C4
132024
60B1
P. riparia
P. asparagi
140647
122924
66D2
47H1
57J3
P. mississippiae
P. ornamentata
402.72
62C7
P. megasperma
P. pinifolia
127953
127952
62B9
55B9
CBS
CH
Isolate identificationb
P. litoralis
P. lacustris
P. gregata
Species
TABLE 1 | Continued
Taiwan
England, UK
Taiwan
Taiwan
France
Taiwan
Indonesia
Japan
India
Japan
The Netherlands
Taiwan
Taiwan
Oregon, USA
1998
n.a.
n.a.
2005
1994
2013
2013
n.a.
1994
1996
Pea
Sweden
2009
Thuja occidentalis North Carolina, 2001 USA
Cucumis sativus
Cajanus cajani
Pueraria lobata
Abies sp.
Stream water
Stream water
Abies sp.
UK
Sweden
Alnus sp. Alnus sp.
Poland
Soil
n.a.
2013
n.a.
2013
2013
1998
2013
1989
MH620066
MH620065
MH620064
MH620063
MH620062
KU681018
KU517150
KU517145
MH620061
KU681017
KU681019
MH620060
DQ674736
MH620059
MH620058
MH620057
MH620056
MH620055
MH620054
HQ261458
MH620053
KC631619
MH620052
2004
2007
MH620051
JN935960
MH620050
KF112860
MH620049
MH620048
JF896561
MH620047
cox1
2006
2007
2012
2012
1931
2008
1972
2009
Year
Michigan, USA 2006
Virginia, USA
Western Australia, Australia
Oregon, USA
Chile
Italy
Mississippi, USA
Washington DC. USA
Western Australia, Australia
England, UK
Western Australia, Australia
Location
Rubus idaeus cv. Scotland, UK Glen Clova
Soil
Fragaria × ananassa
Soil
Soil
Soil
Castanopsis carlesii rhizosphere soil
Syzygium aromaticum
Asparagus officinalis
Irrigation water
Soil
Stream water
Pinus radiata
Soil
Irrigation water
Althaea rosea
Soil
Salix matsudana
Soil
Host or substrate
KT183042
MH620144
KT183041
MH620143
MH620142
AF139368
KU517156
KU517151
KT183037
MH620141
AF139367
MH620140
HQ643340
KU517155
MH620139
KU517153
KU517152
MH620138
KU517154
EF590257
MH620137
n.a.
MH620136
MH620135
MH620134
KP863496
KF112852
MH620133
MH620132
AF266793
MH620131
ITS
KX251735
KX251728
KX251707
KX251686
KX251665
n.a.
KX251644
KX251637
KX251494
KX251588
n.a.
EU080011
KX251564
KX251630
KX251543
KX251623
KX251616
KX251522
KX251609
EU080345
KX251473
KX251375
KX251354
KX251347
KX251333
KX251319
KX251305
KX251285
KX251278
EU080530
KX251250
60S
KX251736
KX251729
KX251708
KX251687
KX251666
KU899239
KX251645
KX251638
KX251495
KX251589
KU899260
EU080012
KX251565
KX251631
KX251544
KX251624
KX251617
KX251523
KX251610
n.a.
KX251474
KX251376
KX251355
KX251348
KX251334
KX251320
KX251306
KX251286
KX251279
EU080531
KX251251
β-tub
KX251737
KX251730
KX251709
KX251688
KX251667
n.a.
KX251646
KX251639
KX251496
KX251590
n.a.
EU080013
KX251566
KX251632
KX251545
KX251625
KX251618
KX251524
KX251611
EU080346
KX251475
KX251377
KX251356
KX251349
KX251335
KX251321
KX251307
KX251287
KX251280
EU080532
KX251252
EF-1α
KX251738
KX251731
KX251710
KX251689
KX251668
n.a.
KX251647
KX251640
KX251497
KX251591
n.a.
KX251571
KX251567
KX251633
KX251546
KX251626
KX251619
KX251525
KX251612
EU080347
KX251476
KX251378
KX251357
KX251350
KX251336
KX251322
KX251308
KX251288
KX251281
EU080533
KX251253
ENL
GenBank accession no.d
KX251739
KX251732
KX251711
KX251690
KX251669
KU899396
KX251648
KX251641
KX251498
KX251592
KU899417
KX251572
KX251568
KX251634
KX251547
KX251627
KX251620
KX251526
KX251613
EU080348
KX251477
KX251379
KX251358
KX251351
KX251337
KX251323
KX251309
KX251289
KX251282
EU080534
KX251254
Hsp90
KX251740
KX251733
KX251712
KX251691
KX251670
n.a.
KX251649
KX251642
KX251499
KX251593
n.a.
EU080015
KX251569
KX251635
KX251548
KX251628
KX251621
KX251527
KX251614
EU080349
KX251478
KX251380
KX251359
KX251352
KX251338
KX251324
KX251310
KX251290
KX251283
EU080535
KX251255
28S
(Continued)
KX251741
KX251734
KX251713
KX251692
KX251671
n.a.
KX251650
KX251643
KX251500
KX251594
n.a.
KX251573
KX251570
KX251636
KX251549
KX251629
KX251622
KX251528
KX251615
EU080350
KX251479
KX251381
KX251360
KX251353
KX251339
KX251325
KX251311
n.a.
KX251284
EU080536
KX251256
tigA
Yang and Hong Evaluating Markers for Identifying Phytophthoras
October 2018 | Volume 9 | Article 2334
Frontiers in Microbiology | www.frontiersin.org
7
8c
8b
8a
7d
7c
(Sub) cladea
121655
270.31
22H1
22H9
32G2
P. hibernalis
P. lateralis
P. ramorum
131246
49J8
61G1
P. taxon parsley MYA-3638 36906
P10974
P7517, P19521
A
MYA-3898
60352
P6871 A
T
A
A
T
T
T
T
A
T
T
T
T
T
T
T
T
T
A
T
T
Indiana, USA
West Virginia, USA
Mississippi, USA
Lactuca sativa
Daucus carota
Oregon, USA
Portugal
Tennessee, USA
Greece
Sweden
n.a.
1931
2004
2006
1995
2013
1997
2001
2009
2004
Camellia japonica South Carolina, n.a. USA
Chamaecyparis lawsoniana
Citrus sinensis
Rhododendron sp.
Petroselinum crispum
Fragaria × ananassa
Japan
Germany
Greece
France
Cichorium intybus The var. foliosum Netherlands
1986
1998
n.a.
n.a.
n.a.
n.a.
Ecuador USA
2006
n.a.
n.a.
Western Australia, Australia
Ohio, USA
Ireland
Brassica oleracea The Netherlands
Abies concolor
Trifolium sp.
Glycine sp.
Zantedeschia aethiopica
Solanum marginatum
Isopogon buxifolius
Medicago sativa
Solanum tuberosum
MH620090
MH620089
MH620088
EU124918
MH620087
MH620086
n.a.
KF358238
MH620085
MH620084
MH620083
MH620082
MH620081
MH620080
MH620079
MH620078
KP288342
KF358236
MH620077
MH620076
MH620074
MH620073
MH620072
MH620071
MH620070
MH620069
MH620068
MH620067
cox1
Beta vulgaris var. California, USA n.a. altissima
1968
2005
2008
2011
1922
n.a.
1959
1986
Year
MH620075
Ireland
Japan
Japan
Virginia, USA
Italy
Indonesia
n.a.
Ontario, Canada
Iran
Location
n.a.
Solanum lycopersicum
Rosa sp.
Fragaria × ananassa
Ilex glabra “Shamrock”
Arbutus unedo
Cinnamomum burmannii
Glycine max
Glycine max
Pistacia vera
Lactuca sativa
P. foliorum
61E7
P. taxon castitis
MYA-4162
MYA-4455
P3103
P1693
P1087
P1738
P2110
T
137103
29E9
P. primulae
P. pseudolactucae
61F4
P. lactucae
34684
60353, 46734 325930
52402
MYA-3900
46724
180615
22938
WPC
Host or substrate
Primula acaulis
127102
61E5
P. dauci
46735 46671
131375
386658
IMI
Typec
620.97
115029
62A8
P. cichorii
24A7
P. sp. kelmania
117687
179.87
62A9
61J8
47H3
P. sansomeana
P. trifolii
240.3
139749
P. brassicae
45F5
P. richardiae
P. pseudocryptogea
P. pseudocryptogea
23A4
129.23
61J2
P. erythroseptica
P. medicaginis
292.35
23J5
P. drechsleri
133248
113.19
61H5
61H9
P. nagaii
P. cryptogea
135747
P. sp. ax
61H4
132772
66C8
46H5
P. parvispora
P. fragariaefolia
144.22
45G6
P. vignae
61J1
16705, MYA-3899
P. cinnamomi
MYA-4082
312.62
22D8
ATCC
33D6
CBS
P. sojae
CH
Isolate identificationb
P. pistaciae
Species
TABLE 1 | Continued
MH620166
MH620165
KT183039
MH620164
MH620163
MH620162
AB894388
KF358226
MH620161
MH620160
MH620159
MH620158
MH620157
MH620156
MH620155
MH620154
KP288376
KF358223
MH620153
MH620152
MH620151
MH620150
MH620149
MH620148
KC478667
MH620147
MH620146
MH620145
KT183043
ITS
KX252147
KX252133
KX252119
KX252112
KX252105
KX252098
n.a.
KX252063
KX252042
KX252014
KX252007
KX252000
KX251986
KX251958
KX251930
KX251923
EU080626
KX251902
KX251895
KX251888
KX251867
KX251860
KX251853
KX251846
KX251839
KX251811
KX251776
KX251762
KX251748
60S
KX252148
KX252134
KX252120
KX252113
KX252106
KX252099
n.a.
KX252064
KX252043
KX252015
KX252008
KX252001
KX251987
KX251959
KX251931
KX251924
EU080627
KX251903
KX251896
KX251889
KX251868
KX251861
KX251854
KX251847
KX251840
KX251812
KX251777
KX251763
KX251749
β-tub
KX252149
KX252135
KX252121
KX252114
KX252107
KX252100
n.a.
KX252065
KX252044
KX252016
KX252009
KX252002
KX251988
KX251960
KX251932
KX251925
EU080628
KX251904
KX251897
KX251890
KX251869
KX251862
KX251855
KX251848
KX251841
KX251813
KX251778
KX251764
KX251750
EF-1α
KX252150
KX252136
KX252122
KX252115
KX252108
KX252101
n.a.
KX252066
KX252045
KX252017
KX252010
KX252003
KX251989
KX251961
KX251933
KX251926
EU080629
KX251905
KX251898
KX251891
KX251870
KX251863
KX251856
KX251849
KX251842
KX251814
KX251779
KX251765
KX251751
ENL
GenBank accession no.d
KX252151
KX252137
KX252123
KX252116
KX252109
KX252102
n.a.
KX252067
KX252046
KX252018
KX252011
KX252004
KX251990
KX251962
KX251934
KX251927
EU080630
KX251906
KX251899
KX251892
KX251871
KX251864
KX251857
KX251850
KX251843
KX251815
KX251780
KX251766
KX251752
Hsp90
KX252152
KX252138
KX252124
KX252117
KX252110
KX252103
n.a.
KX252068
KX252047
KX252019
KX252012
KX252005
KX251991
KX251963
KX251935
KX251928
EU080631
KX251907
KX251900
KX251893
KX251872
KX251865
KX251858
KX251851
KX251844
KX251816
KX251781
KX251767
KX251753
28S
(Continued)
KX252153
KX252139
KX252125
KX252118
KX252111
KX252104
n.a.
KX252069
KX252048
KX252020
KX252013
KX252006
KX251992
KX251964
KX251936
KX251929
n.a.
KX251908
KX251901
KX251894
KX251873
KX251866
KX251859
KX251852
KX251845
KX251817
KX251782
KX251768
KX251754
tigA
Yang and Hong Evaluating Markers for Identifying Phytophthoras
October 2018 | Volume 9 | Article 2334
Frontiers in Microbiology | www.frontiersin.org MYA-4457 MYA-4945
23J7
58A7
47C3
46A2
47C8
P. irrigata
P. macilentosa
P. parsiana
P. virginiana
P. aff. parsiana G1
8
10 MYA-3893 TSD-7
111474
50A1
22G7
45B7
P. gondwanensis
P. intercalaris
140632
291.29
45F9
46J2
P. fallax
125801
38789
46733
P. gallica
55C3
P. constricta
142610
691.79
407.48
P. boehmeriae
46H7
49J9
38E1
46C4
P. captiosa
P. pseudopolonica
P. polonica
9a (cluster P. insolita 9a3)
P. quininea
P. macrochlamydospora-33D5 G2
P. macrochlamydosporaG1 340618
180614
288805
P16826
P6950
P10722
P10719
P15005
T
T
T
T
T
T
A
T
T
Stream water
n.a.
Quercus robur
1994
2005
Year
New Zealand
Western Australia, Australia
New Zealand
Poland
Taiwan
Peru
The Netherlands
New South Wales, Australia
New South Wales, Australia
Ecuador
Ecuador
Iran
Iran
Iran
Iran
Virginia, USA
Iran
Mississippi, USA
Virginia, USA
Virginia, USA
Virginia, USA
Japan
Virginia, USA
Mississippi, USA
Virginia, USA
Ohio, USA
France
2007
n.a.
1998
1927
1997
2006
1992
2006
1980
n.a.
1927
1994
n.a.
n.a.
1993
n.a.
1992
1992
n.a.
2007
1991
2012
2000
2000
2007
2000
2006
2012
California, USA n.a.
Germany
Argentina
Location
Boehmeriae nivea Taiwan
Eucalyptus delegatensis
Soil
Eucalyptus saligna
Soil
Soil
Cinchona officinalis
Zantedeschia aethiopica
Glycine max
n.a.
P10267
T
n.a.
Pistacia vera
Pistacia vera
Pistacia vera
Pistacia vera
Irrigation water
Ficus carica
Irrigation water
Irrigation water
Irrigation water
Irrigation water
Chrysanthemum × morifolium
Irrigation water
Irrigation water
Citrus sp.
Soil
Austrocedrus chilensis
Host or substrate
Glycine max
P8217
P. sp. lagoariana
A
A
A
A
T
T
T
T
T
T
T
T
T
T
T
Typec
P10264
P8213
P8618
P0649
WPC
P. sp. cuyabensis
9a (cluster P. macrochlamydospora-33E1 9a2) G1
9b
60353
47D8
P. aff. parsiana G3
60B5
395328 395330
47C5
P. aff. parsiana G2
395329
IMI
P. aff. parsiana G1
240.3
MYA-4460
05D1
P. hydropathica
MYA-4927
MYA-4919
46A3
P. hydrogena
123163
61F1
MYA-4578
MYA-4944
P. chrysanthemi
58A1
34002
MYA-4074
ATCC
40A6
P. stricta
129273
122911
CBS
Isolate identificationb
9a (cluster P. aquimorbida 9a1)
8
60E9
21H9
41B6
P. obscura
P. austrocedrae
8d
CH
P. syringae
Species
(Sub) cladea
TABLE 1 | Continued
KT163315
KT183046
KF317112
KT183047
KC733451
KC733450
KC733449
n.a.
KC733456
AY564188
MH620099
MH620098
KC733454
MH620097
n.a.
MH620096
MH620095
MH620094
KC295546
KC733455
KF192708
KC733453
KC733452
KC249962
MH620093
GQ294536
KF192702
MH620092
MH620091
KF358233
cox1
KT163268
KT183035
KF317090
KT183036
MH620176
MH620175
MH620174
KY707115
KF358225
GU111612
MH620173
MH620172
KC733445
MH620171
FJ802118
n.a.
MH620170
MH620169
KC295544
KC733446
KF192700
EU334634
EU583793
KC249959
KT183038
FJ666127
KF192694
MH620168
MH620167
KF358220
ITS
KX252610
KX252603
KX252589
EU080161
KX252568
KX252561
EU079658
n.a.
EU080256
EU080175
EU079802
KX252516
KX252510
KX252503
EU080664
KX252463
KX252433
KX252397
KX252378
KX252357
KX252343
KX252315
KX252294
KX252280
KX252266
KX252238
KX252210
KX252196
KX252175
KX252168
60S
KX252611
KX252604
KX252590
EU080162
KX252569
KX252562
EU079659
KY707104
KX252546
EU080176
EU079803
KX252517
KX252511
KX252504
EU080665
KX252464
KX252434
KX252398
KX252379
KX252358
KX252344
KX252316
KX252295
KX252281
KX252267
KX252239
KX252211
KX252197
KX252176
KX252169
β-tub
KX252612
KX252605
KX252591
EU080163
KX252570
KX252563
EU079660
KY787198
EU080258
EU080177
EU079804
KX252518
KX252512
KX252505
EU080666
KX252465
KX252435
KX252399
KX252380
KX252359
KX252345
KX252317
KX252296
KX252282
KX252268
KX252240
KX252212
KX252198
KX252177
KX252170
EF-1α
KX252613
KX252606
KX252592
EU080164
KX252571
KX252564
EU079661
n.a.
EU080259
EU080178
KX252524
n.a.
EU080007
KX252506
EU080667
KX252466
KX252436
KX252400
KX252381
KX252360
KX252346
KX252318
KX252297
KX252283
KX252269
KX252241
KX252213
KX252199
KX252178
KX252171
ENL
GenBank accession no.d
KX252614
KX252607
KX252593
EU080165
KX252572
KX252565
EU079662
n.a.
EU080260
EU080179
EU079805
KX252519
KX252513
KX252507
EU080668
KX252467
KX252437
KX252401
KX252382
KX252361
KX252347
KX252319
KX252298
KX252284
KX252270
KX252242
KX252214
KX252200
KX252179
KX252172
Hsp90
KX252615
KX252608
KX252594
EU080166
KX252573
KX252566
EU079663
n.a.
EU080261
EU080180
EU079806
KX252520
KX252514
KX252508
EU080669
KX252468
KX252438
KX252402
KX252383
KX252362
KX252348
KX252320
KX252299
KX252285
KX252271
KX252243
KX252215
KX252201
KX252180
KX252173
28S
(Continued)
KX252616
KX252609
KX252595
EU080167
KX252574
KX252567
EU079664
n.a.
EU080262
EU080181
EU079807
KX252521
KX252515
KX252509
EU080331
n.a.
n.a.
EU080201
KX252384
KX252363
KX252349
KX252321
KX252300
KX252286
KX252272
KX252244
KX252216
KX252202
KX252181
KX252174
tigA
Yang and Hong Evaluating Markers for Identifying Phytophthoras
October 2018 | Volume 9 | Article 2334
phylogenetic (sub)clade as indicated by the concatenated-sequence tree (TreeBASE S22998). identification: CH, Chuanxue Hong laboratory at Virginia Polytechnic Institute and State University, Virginia Beach, VA, USA; CBS, Centraalbureau voor Schimmelcultures Fungal Biodiversity Centre, Utrecht, The Netherlands; ATCC, American Type Culture Collection, Manassas, VA, USA; IMI, CABI Biosciences, UK; WPC, the World Phytophthora Genetic Resource Collection at University of California, Riverside, USA. c Ex-types (T) or authentic (A) isolates (designated as representative isolates by the originators of the respective species). d Marker: cox1, cytochrome-c oxidase 1 gene; ITS, internal transcribed spacer region; 60S, 60S Ribosomal protein L10; β-tub, beta-tubulin; EF-1α, elongation factor 1 alpha; ENL, enolase; Hsp90, heat shock protein 90; 28S, 28S ribosomal DNA; tigA, tigA gene fusion protein. e n.a., not available.
Pythium aphanidermatum
101728 Outgroup Elongisporangium undulatum
a Molecular
AY564163 337230
32199 60173 135746
357.52 P. sp. boehmeriae-like 45F8
P. lilii n.a.
121982 62B5 P. morindae
46C8 P. kernoviae
Frontiers in Microbiology | www.frontiersin.org
Comparison of Individual-Marker Trees With Concatenated-Sequence Tree Each marker tree for all four selected markers (cox1, ITS, tigA, and β-tub) included a set of identical 150 Phytophthora taxa, plus two outgroup taxa: Elongisporangium undulatum (basionym: Pythium undulatum) was used as the outgroup taxon for ITS, tigA, and β-tub, while Pythium aphanidermatum (Uzuhashi et al., 2010) was used for the mitochondrial marker cox1. The sequence dataset of each marker was aligned in MEGA 7 and edited as described above. Then, the four alignments were combined in MEGA 7 to produce a concatenated sequence alignment. Phylogeny reconstructions including four individualmarker trees and a concatenated-sequence tree were carried out using both Maximum likelihood (ML) and Neighbor joining (NJ) methods with the K2P model and 1000 bootstrap replications in MEGA 7. Alignments and phylogenetic trees have been deposited in TreeBASE (S22998). To validate the accuracy of the concatenated-sequence trees, the clade affiliation of individual species was compared with those presented in previous phylogenetic studies. The overall topological scores between the concatenated-sequence trees and individual-marker trees were calculated using Compare2Trees version September 2011 (Nye et al., 2006).
RESULTS PCR Consistency, Amplifications, and Sequence Alignments The ITS region was the easiest genetic marker to amplify (Table 2). The rates of PCR amplification success for β-tub, 28S, 60S, Hsp90, and EF-1α were also high (>90%; Table 2). Markers tigA, ENL, and cox1 (using primer pair COXF4N and COXR4N) had relatively low success rates (≤80%) with the tigA being the most difficult (Table 2). Sequences could not be obtained from 11 taxa for 60S, 2 for β-tub, 12 for EF-1α, 12 for ENL, 3 for Hsp90, 11 for 28S, 16 for tigA, 2 for ITS, and 6 for cox1 (Table 2). These taxa were excluded from distance analyses of individual markers. Eleven taxa missing any of cox1, ITS, tigA, or β-tub sequences were also excluded in the comparison of individual-marker trees with concatenated-sequence tree. Sequence lengths were consistent for all markers except for the ITS, in spite of missing data at either or both ends of short sequences. Length of ITS sequences varied from 744 bases (P. hydrogena in cluster 9a1 of subclade 9a) to 848 bases (P. intercalaris in clade 10). Among the nine markers, the aligned length was the shortest for 60S and longest for Hsp90 (Table 2). The aligned length of concatenated sequences (cox1, ITS, tigA, and β-tub) of 150 Phytophthora taxa plus the outgroup was 4,714 bases.
Genus-Wide Distance Analyses The mean species distance of cox1 was the highest among the nine markers (Table 2). ITS had the highest genus-wide resolution among the nuclear markers, followed by tigA and βtub (Table 2). ENL, 28S, 60S, Hsp90, and EF-1α had lower species
b Isolate
EU080445 EU080441 FJ802126 1989 Scotland, UK Larix sp. P10342
T
KX252646
AB856800 AB856797
KX252645 KX252644
AB856794 AB856791
KX252643 KX252642
AB856788 AB856782
KX252641 KX252640 KF317089
MG865523
1939
KF317111
Japan
Argentina Citrus sinensis
Lilium sp. T
P1378
A
1987
AB856786
AB856779
KX252639 KX252638 KX252637 KX252636 KX252635 KX252633 MH620178 KT183050 2005 Hawaii, USA Morinda citrifolia var. citrifolia T
P10956
WPC IMI ATCC CH
CBS
Isolate identificationb Species (Sub) cladea
TABLE 1 | Continued
KX252634
KX252631 EU080046 EU080045 EU080044 EU080043 EU080042 MH620177 KT183048
Typec
Host or substrate
Rhododendron ponticum
Location
England, UK
Year
2004
60S ITS
EU080041
ENL EF-1α
Evaluating Markers for Identifying Phytophthoras
cox1
β-tub
GenBank accession no.d
Hsp90
28S
tigA
Yang and Hong
9
October 2018 | Volume 9 | Article 2334
Yang and Hong
Evaluating Markers for Identifying Phytophthoras
TABLE 2 | PCR consistency and overall species distance across the genus Phytophthora by genetic marker. Markera
Internal primers usedb
Aligned length (bases)
No. of amplifications
No. of speciesd
Rate of PCR success (%)c
Mean distancee
Distance range
cox1 ITS
No
867
456
165
75
0.092 ± 0.0003
0–0.256
No
1,039
408
169
99
0.082 ± 0.0003
0–0.182
tigA
Yes
1,670
284
155
71
0.058 ± 0.0001
0–0.110
β-tub
No
1,136
502
169
96
0.043 ± 0.0001
0–0.087
ENL
No
1,168
345
159
80
0.035 ± 0.0001
0–0.097
28S
No
1,273
323
160
97
0.035 ± 0.0002
0–0.088
60S
No
496
344
160
97
0.030 ± 0.0001
0–0.085
Hsp90
Yes
1,758
330
168
95
0.024 ± 0.0001
0–0.116
EF-1α
No
1,015
299
159
98
0.008 ± 0.0000
0–0.024
a Marker: cox1, cytochrome-c oxidase 1 gene; ITS, internal transcribed spacer region; 60S, 60S Ribosomal protein L10; β-tub, beta-tubulin; EF-1α, elongation factor 1 alpha; ENL, enolase; Hsp90, heat shock protein 90; 28S, 28S ribosomal DNA; tigA, tigA gene fusion protein. b Internal primers for sequencing tigA and Hsp90 are listed in Blair et al. (2008). c Rate of successful PCR amplification for each marker done by the authors during the past 6 years. d Number of species (one isolate per species) included in the sequence alignment of each marker. e Overall species distance (mean ± standard error) calculated using the Kimura 2-parameter (K2P) distance model in MEGA 7.
2014; Yang et al., 2017) except that the placement of P. quercina was ambiguous. All trees from sequences of the three nuclear markers had similar topologies (score = 75.3–81.7%) to those of the concatenated-sequences trees in both ML and NJ analyses. In contrast, cox1 sequences produced trees of distinct topologies (TreeBASE S22998). The overall topological similarities to the concatenated-sequences trees were approximately 45% lower than those of nuclear markers in both analyses (Table 5).
distances (mean distance