Open Access

Reliability and Utility of Standard Gene Sequence Barcodes for the Identification and Differentiation of Cyst Nematodes of the Genus Heterodera

Daniel C. Huston
Daniel C. Huston
, 
Manda Khudhir
Manda Khudhir
 and 
Mike Hodda
Mike Hodda
   | Jul 29, 2022
Journal of Nematology's Cover Image
About this article
Previous Article
Next Article
Cite
Article Category: Research Paper
Published Online: Jul 29, 2022
Page range: -
Received: Dec 13, 2021
DOI: https://doi.org/10.2478/jofnem-2022-0024
Keywords
© 2022 Huston et al. published by Sciendo
This work is licensed under the Creative Commons Attribution 4.0 International License.

Figure 1

Geographic origin of 18S, ITS, 28S, and coxI gene sequences analyzed in the present study. The size of the circle reflects the total number of sequences of all markers centered on the country of origin.
Geographic origin of 18S, ITS, 28S, and coxI gene sequences analyzed in the present study. The size of the circle reflects the total number of sequences of all markers centered on the country of origin.

Figure 2

Neighbor-joining tree based on coxI sequences of Heterodera avenae from the NCBI database GenBank showing a clade of mislabeled sequences.
Neighbor-joining tree based on coxI sequences of Heterodera avenae from the NCBI database GenBank showing a clade of mislabeled sequences.

Figure 3

Neighbor joining (A) and maximum likelihood (B) trees derived from analyses of ITS gene sequences for the Schachtii species group showing multiple instances where species cannot be differentiated.
Neighbor joining (A) and maximum likelihood (B) trees derived from analyses of ITS gene sequences for the Schachtii species group showing multiple instances where species cannot be differentiated.

Species pairs of Heterodera which are inadequately or poorly distinguished from one another using one or more of the standard molecular markers evaluated (18S, ITS, 28S, coxI), including minimum base pair differences observed between sequences of problematic species pairs.

Species group Species pair Gene region Minimum base pair difference Delineation power Notes
Avenae H. avenae–H. filipjevi 18S 1 Inadequate
Avenae H. avenae–H. hordecalis 18S 4 Weak H. hordecalis not monophyletic
in NJ/ML analyses; potentially
distinguishable from closely
related species in isolated
analyses.
Avenae H. avenae–H. mani 18S 1 Inadequate
Avenae H. filipjevi–H. hordecalis 18S 1 Inadequate
Avenae H. filipjevi–H. mani 18S 2 Inadequate
Avenae H. hordecalis–H. mani 18S 3 Weak See above note.
Avenae H. arenaria–H. avenae ITS 0 Inadequate
Avenae H. avenae–H. pratensis ITS 0 Inadequate
Avenae H. avenae–H. australis ITS 2 Weak Although only two bp different,
sequences of H. australis form
clade with some sequences
of “H. avenae” which have
been shown to be H. australis
(see Subbotin et al., 2018).
Avenae H. avenae–H. mani ITS 2 Weak Despite only two bp difference,
sequences of H. mani form
monophyletic clade to
exclusion of other closely
related sequences.
Avenae H. aucklandica–H. 28S 1 Inadequate Only one 28S sequence of
avenae H. aucklandica available for
comparison.
Avenae H. aucklandica–H. 28S 1 Inadequate Two 28S sequences of
hordecalis H. hordecalis 9 bp different
Avenae H. aucklandica–H. 28S 1 Inadequate Only one 28S sequence of
pratensis H. pratensis available for
comparison.
Avenae H. avenae–H. pratensis 28S 0 inadequate See above note.
Avenae H. avenae–H. hordecalis 28S 0 Inadequate Two 28S sequences of
H. hordecalis 9 bp different
Avenae H. arenaria–H. avenae cox1 2 Weak Despite only two bp
difference, H. arenaria forms
monophyletic clade within
H. avenae group.
Cyperi H. elachista–H. oryzae ITS 1 Inadequate
Cyperi H. elachista–H. oryzae 28S 0 Inadequate
Goettingiana H. carotae–H. cruciferae ITS 1 Inadequate
Goettingiana H. goettingiana–H. 28S 0 Inadequate Two 28S sequences of
microulae H. goettingiana are 19 bp
different from one another; one
potentially misidentified
Goettingiana H. carotae–H. urticae 28S 2 Weak Only one 28S sequence
of H. urticae available for
comparison.
Goettingiana H. carotae–H. cruciferae 28S 2 Weak Only one 28S sequence of
H. cruciferae available for
comparison (two on GenBank
but one appears to actually be
H. schachtii).
Goettingiana H. carotae–H. cruciferae cox1 1 Inadequate Three cox1 sequences
available for H. cruciferae, one
15 bp different from others.
Schachtii H. betae–H. glycines 18S 2 Weak H. glycines forms
monophyletic clade to
exclusion of H. betae,
H. schachtii & H. trifolii in ML,
but not NJ, analyses.
Schachtii H. betae–H. schachtii 18S 0 Inadequate
Schachtii H. betae–H. trifolii 18S 0 Inadequate
Schachtii H. schachtii–H. glycines 18S 2 Weak See above note.
Schachtii H. schachtii–H. trifolii 18S 0 Inadequate
Schachtii H. trifolii–H. glycines 18S 2 Weak See above note.
Schachtii H. betae–H. daverti ITS 0 Inadequate
Schachtii H. betae–H. schachtii ITS 0 Inadequate
Schachtii H. betae–H. trifolii ITS 0 Inadequate
Schachtii H. ciceri–H. schachtii ITS 2 Inadequate
Schachtii H. ciceri–H. trifolii ITS 1 Inadequate
Schachtii H. daverti–H. schacthii ITS 0 Inadequate
Schachtii H. daverti–H. trifolii ITS 0 Inadequate
Schachtii H. glycines–H. ITS 1 Inadequate
medicaginis
Schachtii H. schachtii–H. trifolii ITS 0 Inadequate
Schachtii H. betae–H. daverti 28S 1 Inadequate
Schachtii H. betae–H. schachtii 28S 2 Inadequate
Schachtii H. betae–H. trifolii 28S 1 Inadequate
Schachtii H. daverti–H. schachtii 28S 3 Weak
Schachtii H. daverti–H. trifolii 28S 1 Inadequate
Schachtii H. schachtii–H. trifolii 28S 1 Inadequate

Numbers of species of Heterodera for which one or more gene sequences of one or more molecular markers (18S, ITS, 28S, and coxI) are available on GenBank.

Marker
Species of Heterodera 18S ITS 28S coxI All
Species with at least one sequence 18 55 36 38 13
Species with more than one sequence 13 38 22 29 11

Species of Heterodera, and associated number of sequences, recorded from 59 countries based on data from the NCBI database GenBank, up to 10 August 2021.

Total number of sequences recorded
Species Country 18S ITS 28S coxI
Heterodera arenaria Italy 1 3
Netherlands 2
United Kingdom 3 1
Heterodera aucklandica Belgium 1
New Zealand 1 1 3
United Kingdom 1 2
Heterodera australis Australia 7 5
China 11
Heterodera avenae Algeria 4 6
Australia 1
Azerbaijan 9
China 4 359 57 4
Czech Republic 7 7 7
Egypt 5 3
France 9 7
Germany 3 4
India 4 1
Iran 2 1 4
Iraq 3
Israel 3 4
Morocco 27 5
Norway 4
Pakistan 1
Saudi Arabia 23 1 1
Serbia 1 1
South Korea 1 2 1
Spain 3 3
Switzerland 1
Syria 3 6
Tunisia 1 6
Turkey 39 26
United Kingdom 1
USA 1 10 3 14
Unknown 4
Heterodera betae Belgium 1
Germany 3
Netherlands 3 2 2
Unknown 2
Heterodera bifenestra Belgium 2
Sweden 1 1
Heterodera cajani India 12 13
Heterodera cardiolata Pakistan 1 1
United Kingdom 1
Heterodera carotae Belgium 1
Algeria 2
Canada 5 6 5
France 1 2
Italy 3 1 5
Mexico 2 1
South Africa 1 1 1
Switzerland 1
Heterodera ciceri Syria 3 1
Heterodera circeae Germany 1
Heterodera cruciferae Algeria 5
Iran 1 1
Italy 1 1
Netherlands 1
Russia 1 1
Serbia 1
USA 1 2
Heterodera cyperi Spain 1
USA 1 1
Heterodera daverti Germany 3 2
Italy 1 1 1
Netherlands 1 1
Heterodera dunensis Spain 3 3 3 8
Heterodera elachista China 1 27 10
Iran 2 1 1
Italy 2 4 4 2
Japan 11 3
Heterodera fici Canada 2
Georgia 1 1
Greece 4
Iran 1 3
Italy 2 1
Qatar 1
USA 2 1 1
Heterodera filipjevi Algeria 2
Azerbaijan 20
China 58 7 2
Germany 2 1
Greece 1
India 1
Iran 5 19
Italy 1
Kazakhstan 25
Kyrgyzstan 31
Russia 3 7
Serbia 1 2
Slovakia 1
Spain 1 2
Sweden 1 1
Syria 6
Tajikistan 1 3
Turkey 80 20
Ukraine 3
United Kingdom 1 3
USA 3 1 5
Unknown 2
Heterodera glycines Canada 1 12 1
Chile 1
China 5 275 29
Iran 2 1
Japan 6 10
South Korea USA 1 4 21 7 7 81
Unknown 1 1
Heterodera goettingiana China 1 10 1 3
Germany 2 1
Iran 1
Ireland 1
Italy 2
Netherlands 4
USA 1
Heterodera goldeni Egypt 2
Iran 2
Israel 1
Heterodera graminis India 1
Heterodera graminophila USA 1
Heterodera guangdongensis China 3 3
Myanmar 2
Vietnam 2 1
Heterodera hainanensis China 1 1
Heterodera hordecalis Algeria 2 21 2
Estonia 1
Germany 1 2
Iran 4 3
Israel 3 2
Italy 2
Netherlands 1 2
Slovakia 1 2
Sweden 1 1
Tunisia 1
United Kingdom 1
Heterodera humuli Belgium 1
Germany 1 2
Iran 3 1 8
Kyrgyzstan 1 1
Portugal 1
Russia 1 2
USA 6 5
Heterodera koreana China 1 2 2
Iran 1 1
Japan 20 41 41
South Korea 3 3 3
USA 2 3 1
Heterodera latipons Cyprus 21
Iran 3 3
Israel 2
Jordan 2 3
Kazakhstan 28
Morocco 3
Russia 1 1
Syria 9 1 7
Turkey 87 3
Heterodera litoralis New Zealand 1 1 4
Heterodera mani Germany 4 1
United Kingdom 1
USA 1 4
Unknown 1
Heterodera medicaginis Russia 1
USA 13 1 14
Heterodera mediterranea Italy 2 2
Spain 2
Tunisia 1 2 2
Heterodera microulae China 1 1 1
Heterodera mothi Azerbaijan 1 1
Iran 1 1
Heterodera orientalis Guatemala 2
Russia 1
USA 2 1
Heterodera oryzae South Korea 1 1 1
Heterodera oryzicola India 1 1
Heterodera persica Iran 1
Heterodera pratensis Belgium 1
Germany 8 8
Iran 5
Netherlands 1 1
Russia 1 2
South Korea 2 1 2
USA 1 1
Heterodera ripae Belgium Germany 1 5 4
Greece 1 2
Russia Serbia 1 1 4
Slovakia 2
Sweden 1
United Kingdom 2
Heterodera sacchari Ghana 2
Ivory Coast 1
Heterodera salixophila Belgium 1 1 3
Germany 4
Iran 1 1
Russia 5
Ukraine 3 4
Heterodera schachtii Algeria 1
Australia 4
Belgium 1 15
China 1 1 8 3
France 1 3
Germany 4
Iran 2 1
Ireland 2
Japan 1 1 1
Mexico 1 2
Morocco 3
Netherlands 8 1 1
Poland 1 1
Serbia 3
South Africa 1
South Korea 3 4 7
Turkey 12
USA 2 3 22
Unknown 6 1
Heterodera scutellariae Germany 1
Heterodera sinensis China 1
Heterodera skohensis India 1
Heterodera sojae China 1 1
Japan 1 1
South Korea 3 8 12
Heterodera sorghi India 1
Heterodera sturhani China 7
Heterodera trifolii Costa Rica 1 1 1
Estonia 1
Iran 1 1 2
Italy 1
Japan 8 8 8
South Korea 8 8 9
United Kingdom 1 2 1
USA 2 5
Unknown 4
Heterodera turcomanica Iran 1 1
Heterodera urticae Belgium 1 1 3
Heterodera ustinovi Belgium 1
Germany 1
Slovakia 1
United Kingdom 1
USA 1 2
Heterodera vallicola Russia 1 1
Heterodera zeae Afghanistan 1
China 5
Greece 1 5 1
India 4
Portugal 1
USA 2 1

Total number of sequences of species of Heterodera per country available on the NCBI database GenBank, up to 10 August 2021.

Number of sequences recorded
ITS 18S 28S coxI Total
Afghanistan 1 1
Algeria 62 7 8 77
Australia 11 1 6 18
Azerbaijan 36 1 37
Belgium 21 1 2 14 38
Canada 19 1 7 5 32
Chile 1 1
China 761 16 131 21 937
Costa Rica 1 1 1 3
Cyprus 21 21
Czech Republic 7 7 7 21
Egypt 7 3 10
Estonia 2 2
France 11 9 20
Georgia 1 1 2
Germany 35 1 28 64
Ghana 2 2
Greece 6 1 1 7 15
Guatemala 2 2
India 26 15 1 42
Iran 41 16 51 108
Iraq 3 8 11
Ireland 1 2 3
Israel 7 7
Italy 19 6 8 12 45
Ivory Coast 1 1
Japan 47 64 50 161
Jordan 2 3 5
Kazakhstan 53 53
Kyrgyzstan 32 1 33
Madagascar 2 2
Mexico 3 3 6
Morocco 34 5 39
Myanmar 2 2
Netherlands 16 2 3 7 28
New Zealand 2 2 7 11
Norway 4 4
Pakistan 2 1 3
Poland 1 2 3
Portugal 1 1 2
Qatar 1 1
Russia 12 23 35
Saudi Arabia 23 1 1 25
Serbia 7 3 10
Slovak Republic 3 4 7
South Africa 3 1 1 5
South Korea 30 38 36 104
Spain 10 3 3 13 29
Sweden 4 2 2 8
Switzerland 3 3
Syria 15 1 21 37
Tajikistan 1 3 4
Tunisia 4 1 2 6 13
Turkey 219 53 272
Ukraine 3 1 7 11
United Kingdom 11 1 9 21
USA 71 7 18 158 254
Vietnam 2 1 3
Unknown 2 21 1 1 25
Total 1724 77 346 592 2739

Intraspecific differences observed in the 18S, ITS, 28S, and coxl gene regions for those species of Heterodera for which more than one sequence of one or more of the respective gene regions are available, excluding problematic sequences.

18S ITS 28S cox1
Species n bp (%) n bp (%) n bp (%) n bp (%)
Heterodera arenaria 4 0–1; 0.50 (0–0.29; 0.15) 6 0–2; 2 (0–0.48; 0.48)
Heterodera aucklandica 3 0(0) 5 0–1; 0 (0–0.24; 0)
Heterodera australis 18 0–9; 3 (0–0.93; 0.31) 5 0(0)
Heterodera avenae 17 0–11; 1 (0–1.09; 0.06) 542 0–80; 8 (0–11.89; 0.89) 76 0–5; 0 (0–0.67; 0) 91 0–34; 9 (0–8.17; 2.40)
Heterodera betae 2 1 (0.06) 7 0–12; 3 (0–1.29; 0.37) 2 0(0) 2 0(0)
Heterodera bifenestra 3 2–10; 8 (0.20–1.02; 0.81)
Heterodera cajani 12 0–12; 4 (0–1.18; 0.39) 13 0–11; 2 (0–1.46; 0.26)
Heterodera carotae 13 0–13; 6 (0–1.35; 0.62) 8 0–6; 3 (0–0.83; 0.41) 17 0–20; 3 (0–4.77; 0.82)
Heterodera ciceri 3 7–14; 11 (0.74–1.49; 1.18)
Heterodera cruciferae 11 0–10; 3 (0–1.21; 0.36) 2 52 (9.49) 3 1–16; 16 (0.24–4.01; 3.77)
Heterodera cyperi 2 0(0)
Heterodera daverti 5 0–2; 1 (0–0.24; 0.11) 3 0–2; 2 (0–0.51; 0.51)
Heterodera dunensis 3 0(0) 3 0(0) 3 0(0) 8 0–1; 0 (0–0.28; 0)
Heterodera elachista 3 3–4; 3 (0.25–0.36; 0.36) 43 0–48; 11 (0–4.52; 1.05) 18 0–4; 1 (0–0.54; 0.15) 2 0(0)
Heterodera fici 2 2 (0.34) 8 0–3; 1 (0–0.31; 0.10) 9 0–2; 1 (0–0.47; 0.24)
Heterodera filipjevi 3 1–2; 1 (0.12–0.17; 0.17) 256 0–43; 2 (0–4.46; 0.21) 10 0–2; 1 (0–0.30; 0.13) 75 0–44; 10 (0–10.58; 2.42)
Heterodera glycines 6 0–1; 0 (0–0.16; 0) 319 0–112; 3 (0–11.89; 0.32) 56 0–43; 2 (0–5.79; 0.26) 81 0–9; 0 (0–1.04; 0)
Heterodera goettingiana 4 0–10; 5 (0–1.21; 0.59) 15 0–81; 37 (0–11.34; 5.08) 2 19 (2.92) 3 0–17; 17 (0–4.84; 4.84)
Heterodera goldeni 5 0–6; 2 (0–0.59; 0.23)
Heterodera guangdongensis 3 0–3; 3 (0–0.26; 0.26) 7 1–6; 4 (0.15–0.91; 0.58)
Heterodera hordecalis 2 5 (0.31) 37 0–68; 7 (0–8.89; 0.84) 2 9 (1.33) 10 0–47; 14 (0–11.4; 3.37)
Heterodera humuli 13 0–4; 1 (0–0.57; 0.17) 19 0–9; 2 (0–5.92; 0.59)
Heterodera koreana 3 1–22; 2 (0.17–1.27; 0.34) 29 0–28; 7 (0–3.08; 0.73) 48 0–21; 2 (0–2.88; 0.27) 44 0–18; 5 (0–4.57; 1.27)
Heterodera latipons 154 0–122; 13 (0–13.32; 1.46) 2 1 (0.15) 16 0–50; 27 (0–12.05; 6.52)
Heterodera litoralis 4 0(0)
Heterodera mani 6 1–9; 3 (0.10–0.93; 0.31) 6 0–1; 1 (0–0.27; 0.24)
Heterodera medicaginis 14 0–9; 3 (0–0.95; 0.42) 14 0–2; 0 (0–0.49; 0)
Heterodera mediterranea 2 1 (0.17) 6 1–9; 7 (0.11–0.95; 0.74) 2 3 (0.38)
Heterodera mothi 2 3 (0.30) 2 2 (0.44)
Heterodera orientalis 5 6–18; 12 (0.66–1.94; 1.28)
Heterodera pratensis 13 0–10; 6 (0–1.08; 0.62) 20 0–8; 2 (0–1.93; 0.53)
Heterodera ripae 5 0–4; 2 (0–0.46; 0.22) 19 0–11; 1 (0–2.59; 0.24)
Heterodera sacchari 3 0–1; 1 (0–0.09; 0.09)
Heterodera salixophila 5 0–20; 15 (0–2.10; 1.58) 2 1 (0.15) 16 0–18; 2 (0–4.57; 0.51)
Heterodera schachtii 9 1–9; 4 (0.06–0.99; 0.31) 64 0–31; 12 (0–3.04; 1.29) 20 0–4; 1 (0–0.58; 0.13) 38 0–7; 1 (0–1.88; 0.12)
Heterodera sojae 5 0–12; 4.5 (0–1.36; 1.03) 10 0–8; 4 (0–1.06; 0.53) 12 0–6; 1 (0–1.59; 0.24)
Heterodera sturhani 7 0–3; 0 (0–0.72; 0)
Heterodera trifolii 5 0–8; 3 (0–0.47; 0.29) 23 0–14; 3 (0–1.48; 0.43) 19 0–1; 0 (0–0.15; 0) 26 0–5; 0 (0–1.24; 0)
Heterodera ustinovi 5 1–8; 3.5 (0.10–0.88; 0.36) 5 0–1; 1 (0–0.24; 0.24)
Heterodera zeae 16 0–39; 14 (0–5.65; 2.19) 4 0–2; 1 (0–0.31; 0.15)
eISSN:
2640-396X
Language:
English
Publication timeframe:
Volume Open
Journal Subjects:
Life Sciences, other
Journal RSS Feed