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8Russian Journal of Nematology, 2021, 29 (2), 93 - 100
DNA barcoding and phylogeny of some cyst nematode species from the genus Heterodera (Tylenchida: Heteroderidae) parasitising monocots
Sergei A. Subbotin1, 2, Antoinette Swart3, 4, Ignacio Cid del Prado Vera5, Zahra Tanha
Maafi6 and Vladimir N. Chizhov2
'Plant Pest Diagnostic Center, California Department of Food and Agriculture, 3294 Meadowview Road,
95832-1448, Sacramento, CA, USA e-mail: sergei. a. subbotin@gmail. com 2A.N. Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, Leninskii Prospect 33,
119071, Moscow, Russia
3Nematology Unit, Biosystematic Division, ARC-Plant Protection Research Institute, Private Bag X134,
0121, Queenswood, Pretoria, South Africa ^Department of Zoology, University of Johannesburg, Private Bag 524, Auckland Park, 2006,
Johannesburg, South Africa 5Colegio de Postgraduados, 56230, Montecillo, Mexico 6Iranian Research Institute of Plant Protection, Agricultural Research Education and Extension Organization (AREEO), Tehran, Iran
Accepted for publication 01June 2021
Summary. The genus Heterodera presently contains 87 valid species, of which five species: H. bifenestra, H. cardiolata, H. cyperi, H. goldeni and H. sacchari are molecularly characterised in this study. Using molecular criteria, we also distinguished five putative new species: Heterodera sp. 1-sp. 5 from the Afenestrata, Cardiolata, Bifenestra and Sacchari groups. A total of 16 new ITS rRNA, four new D2-D3 expansion segments of 28S rRNA and 23 new partial COI gene sequences were obtained from 14 cyst nematode populations collected from nine countries: Ghana, Iran, Mexico, Pakistan, Russia, Spain, Tanzania, The Netherlands and Ukraine. Phylogenetic relationships within the genus Heterodera are presented using these three gene fragments. The study confirmed the conclusions that each cyst nematode species has a unique COI sequence or DNA barcode that enables its identification and separation from all other species.
Key words: 28S rRNA gene; COI gene; ITS rRNA gene; Mexico, molecular phylogeny; Russia, Tanzania.
Presently, the genus Heterodera contains 87 valid species of cyst nematodes (Handoo & Subbotin, 2018; Li et al., 2020; Singh et al., 2020). This genus has been divided by using morphological and molecular characteristics into nine species groups: Afenestrata, Avenae, Bifenestra, Cardiolata, Cyperi, Goettingiana, Humuli, Sacchari and Schachtii (Handoo & Subbotin, 2018). Representatives of six groups (Afenestrata, Avenae, Bifenestra, Cardiolata, Cyperi and Sacchari) parasitise monocotyledons. Recently, the mitochondrial COI gene has also been successfully applied for the identification and DNA barcoding of species from the Avenae group (Subbotin et al., 2018) and Schachtii group (Powers et al., 2019); however, this gene was not used for characterisation of species from Bifenestra and Sacchari groups.
The main goal of this study was the molecular characterisation of some cyst nematode species parasitising monocotyledons using sequences of rRNA and COI gene fragments. The nematode samples used for this analysis were collected during surveys in several countries.
MATERIAL AND METHODS
Nematode samples. Species and populations from different hosts and localities used in this study are listed in Table 1. A total of 14 nematode populations collected in nine countries were analysed in this study. Several valid and unidentified species characterised in the previous studies (Subbotin et al., 2001; Tanha Maafi et al., 2003, 2007; Mundo-Ocampo et al., 2008) were also
© Russian Society of Nematologists, 2021; doi: 10.24412/0869-6918-2021-2-93-100
included. Cysts from Tanzania were collected from just north of Bagamoya (north of Dar Es Salaam), on a cattle farm (Razaba Farm) in natural vegetation (92°92'5.51" N; 47°70'3.2" E) by Dr Vaughan Spaull on 6 October 2008. Cysts from unknown
plants were collected in Mexico in two locations (Table 1). Cysts were extracted from soil samples using standard flotation and sieving techniques. Species identification of the studied populations was accomplished by integrating results of morphological
Fig. 1. Phylogenetic relationships between some cyst nematode species as inferred from Bayesian analysis of the ITS rRNA gene sequences under the GTR + I + G model. Posterior probability values are given for appropriate clades. New sequences are indicated by bold font. * - originally identified as Heterodera graminis in the GenBank. Group names are given for appropriate species.
Table 1. Species and populations of cyst nematodes of the genus Heterodera parasitising monocots used in the present study.
vo
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Species Location Host Sample code D2-D3 of 28S rRNA gene GenBank accession number COI gene GenBank accession number ITS rRNA gene GenBank accession number Source and/or reference
H. cyperi Spain Cvperus sp. CD2946 - MW374286, MW374287 AF274388 M. Romero, Subbotin et al. (2001)
H. cardiolata Pakistan Cvnodon dactvlon CD274, CD2967 DQ328698 MW374265, MW374266 AF274386 F. Shahina, Subbotin et al. (2001, 2006)
H. bifenestra The Netherlands Grasses 559 - MW374275 - S.A. Subbotin
H. goldeni Iran, Gilan province, Bandar-e Anzali Phragmites australis Zah20 - MW374278, MW374279 - Z. Tanlia Maafi, Tanlia Maafi et al. (2003)
H. goldeni Iran, Mazandaran province, Nashtarud P. australis Zah23 - MW3 74282 AF498383 Z. Tanlia Maafi, Tanlia Maafi et al. (2003)
H. goldeni Iran, Gilan province P. australis 840, 3Gi - MW3 74280, MW3 74281, MW3 74283 EF143610 Z. Tanlia Maafi, Tanlia Maafi et al. (2007)
H. sacchari Ghana, Ashani region Oryza sativa CD2960 - MW3 74284 EF143604, EF143605 D. Hunt, Tanlia Maafi et al. (2007)
Heterodera sp. 1 Tanzania, Bagamoya Unknown plant CD550 MW3 70469, MW3 70470 MW374285 MW3 70471 A. Swart
Heterodera sp. 2 Mexico, Michoacan, Cumuatillo Unknown plant CD 522 - MW3 74274 MW3 70476 M. Mundo-Ocampo
Heterodera sp. 2 Mexico, Hidalgo, Singuilucan, Rincon del Puerto, Sample 75, trip 2 Unknown plant CD3011 - MW3 74270-MW3 74273 MW3 70477-MW370485 I. Cid del Prado Vera
Heterodera sp. 3 Russia, Kostroma region Grasses CD 562 - MW3 74276 MW370474, MW370475 V.N. Cliizhov
Heterodera sp. 4 Ukraine, Kherson C. dactvlon CD273 EU284031 - MW3 70472, MW3 70473 S.A. Subbotin, Mundo-Ocampo et al. (2008)
Heterodera sp. 4 Iran, Ardabil, Moghan Phragmites sp. Zah24 - MW3 74267-MW3 74269 AF498373 Z. Tanlia Maafi, Tanlia Maafi et al. (2003)
Heterodera sp. 5 Tanzania, Bagamoya Unknown plant CD551 MW3 70467, MW3 70468 MW374277 MW370486 A. Swart
g
o*
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>! o
S-a
o §
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Fig. 2. Phylogenetic relationships between some cyst nematode species as inferred from Bayesian analysis of the D2-D3 of 28 S rRNA gene sequences under the GTR + I + G model. Posterior probability values are given for appropriate clades. New sequences are indicated by bold font. Group names are given for appropriate species.
and morphometric studies, and phylogenetic and sequence analysis (Subbotin et al., 2010a). For scanning electron microscopy (SEM) of the vulval cone, cysts were prepared following the technique of Lax and Doucet (2002), after which they were mounted on microscope stubs and coated with goldpalladium (21 nm). The cysts were examined with a FEI Quanta FEG 250 scanning electron microscope at 5-10 kV. Photographs were taken by L.R. Tiedt (Laboratory for Electron Microscopy, North West University, Potchefstroom, South Africa).
DNA extraction, PCR and sequencing. DNA was extracted from 50 or more J2 and embryonated eggs released from single cysts. DNA extraction, PCR, cloning and sequencing were made as described in Tanha Maafi et al. (2003) and Subbotin
et al. (2018). Several primer sets were used in the present study: forward TW81 (5'-GTT TCC GTA GGT GAA CCT GC-3') and reverse AB28 (5'-ATA TGC TTA AGT TCA GCG GGT-3') for amplification of the ITS1-5.8S-ITS2 rRNA gene; forward D2A (5'-ACA AGT ACC GT GAG GGA AAG TTG-3') and reverse D3B (5'-TCG GAA GGA ACC AGC TAC TA-3') for amplification of the D2-D3 of 28S rRNA gene and forward Het-coxiF (5'-TAG TTG ATC GTA ATT TTA ATG G-3') and reverse Het-coxiR (5'-CCT AAA AC A TAA TGA AAA TGW GC-3') for amplification of part of the COI gene. Sequencing was performed by Genewiz Inc. (California, USA). New sequences were deposited in the GenBank database under accession numbers MW370471-MW370486 (ITS
rRNA gene); MW370467-MW370470 (D2-D3 of 28S rRNA gene); MW374265-MW374287 (COI gene).
Phylogenetic analysis. Alignments with the ITS rRNA, the D2-D3 of 28S rRNA and COI gene sequences were generated using ClustalX 1.83 (Chenna et al., 2003) with default parameters. New sequences were aligned with corresponding published gene sequences (Subbotin et al., 2001;
Phylogeny of Heterodera from monocots
Tanha Maafi et al., 2003; Sekimoto et al., 2017; Li et al., 2020; Singh et al., 2020 and others). The sequence alignments were analysed with Bayesian inference (BI) using MrBayes 3.1.2 (Ronquist & Huelsenbeck, 2003) as described by Subbotin et al. (2018). The best fit models of DNA evolution were obtained using the program jModeltest. 0.1.1 (Posada, 2008) with the Akaike Information Criterion.
H. cyperi ( MW374286, CD2946a, Spain) H. cyperi ( MW374287, CD2946b, Spain)
H. cyperi (MG857126, USA, Georgia) -H. mothi (MH144208)
H. guangdongensis (mf425735)
J '
100 R I
H. oryzae (mn720173) H. oryzae (mt823012) H. elachista (mh144207)
- H. sojae (mn720177) H. goldeni (MW374278, Zah20-2, Iran) H. goldeni (MW374279, Zah20-3, Iran) H. goldeni (MW374280, 840a 3GI, Iran) H. goldeni (MW374283, 840c, Iran) H. goldeni (MW374282, Za23-3, Iran) H. goldeni (MW374281, 840b 3GI, Iran) H. sacchari (MW374284, CD2960b, Ghana) Heterodera sp.1 (MW374285, CD550, Tanzania)
so |-H. mani (mg523095)
ü| I-H. avenae (mg522984)
l— H f
Cyperi
Sacchari
H. australis (KU 147202)
H. ustinovi (MG523090)
WC
Avenae
ci
— H. hordecalis (mg523145)
— H. filipjevi (mg523074) H. latipons (mg523127)
H. glycines (mk093121) H. medicaginis (mk093170) SchachtH
H. schachtii (mk093067) H. trifolii (mn720174)
Heterodera sp.2 (MW374270, CD3011c, Mexico) Heterodera sp.2 (MW374272, CD3011p, Mexico) p} Heterodera sp.2 (MW374271, CD3011d, Mexico) Heterodera sp.2 (MW374273, CD3011f, Mexico) L- Heterodera sp.2 (MW374274, CD522, Mexico)
100.-H. bifenestra (MW374275, 559, The Netherlands)
I-Heterodera sp.3 (MW374276, CD562e, Russia)
H. cardiolata (MW374265, CD274, Pakistan) H. cardiolata (MW374266, CD2967a, Pakistan) H. cardiolata (hm640929, Pakistan) Heterodera sp.4 (MW374267, Zah24-8, Iran) Heterodera sp.4 (MW374268, Zah24-9, Iran) Heterodera sp.4 (MW374269, Zah24-4, Iran) H. koreana (lc202190)
-Heterodera sp.5 (MW374277, CD551, Tanzania)
-H. cruciferae (mg563230) i
— h. goettingiana (kyi 29830) i Goettinqiana
H. microulae (mt576084) |
Bifenestra
Cardiolata
Afenestrata
— Meloidodera mexicana (MF425726) — Meloidodera astonei {MF425727)
0.1
Fig. 3. Phylogenetic relationships between some cyst nematode species as inferred from Bayesian analysis of the COI mtDNA gene sequences under the GTR + I + G model. Posterior probability values are given for appropriate clades. New sequences are indicated by bold font. Group names are given for appropriate species.
RESULTS AND DISCUSSION
A total of 16 new ITS rRNA, four new D2-D3 of 28S rRNA and 23 new COI gene sequences were obtained in this study from 14 cyst nematode samples. Five valid species: H. bifenestra Cooper, 1955; H. cardiolata Kirjanova & Ivanova, 1969; H. cyperi Golden, Rau & Cobb, 1962; H. goldeni Handoo & Ibrahim, 2002 and H. sacchari Luc & Merny, 1963 were molecularly characterised. Using molecular criteria, we distinguished five putative new species: Heterodera sp. 1 from the Sacchari group, Heterodera sp. 2 and Heterodera sp. 3 from the Bifenestra group, Heterodera sp. 4 from the Cardiolata group, and Heterodera sp. 5 from the Afenestrata group. Heterodera sp. 2 was not assigned to any of the groups because information on morphological characteristics was lacking. Plant hosts for Heterodera sp. 1, sp. 2 and sp. 5 were not identified; however, it has been assumed that they might belong to monocots.
The ITS rRNA gene sequence alignment contained 70 sequences of Heterodera, and four sequences of outgroup taxa and was 1243 bp in length. Phylogenetic relationships between Heterodera species are given in Figure 1 and they are congruent with those published by Subbotin et al. (2001) and Tanha Maafi et al. (2003). Interspecific variation between H. bifenestra and Heterodera sp. 3 was 3.5-3.7% (34-36 bp), between H. cardiolata and Heterodera sp. 4 it was 2.3-3.4%
(22-32 bp) and between H. orientalis and Heterodera sp. 5 it was 6.8% (63 bp).
The D2-D3 of 28S rRNA gene sequence alignment contained 29 sequences of Heterodera, and two sequences of outgroup taxa and was 719 bp in length. Phylogenetic relationships between Heterodera species are given in Figure 2. Interspecific variation between H. orientalis and Heterodera sp. 5 was 3.0-3.1% (20-21 bp).
The COI gene sequence alignment contained 46 sequences of Heterodera, and two sequences of outgroup taxa and was 441 bp in length. The phylogenetic relationships are given in Figure 3. Interspecific variation between H. bifenestra and Heterodera sp. 3 was 9.3% (40 bp), between H. cardiolata and Heterodera sp. 4 it was 5.3-6.1% (23-26 bp) and between H. glodeni and H. sacchari it was 5.2-5.4% (23-24 bp).
Heterodera bifenestra is a widely distributed species in grassland in Europe. This species is characterised by bifenestrate cysts. Subbotin et al. (2010b) reported that RFLP-ITS-PCR with the restriction enzyme MvaI distinguishes H. bifenestra from morphologically similar putative new sibling species Heterodera sp. 3 found in Russia. In this study, we provided the ITS rRNA and COI sequences of this putative new species for the first time. Heterodera bifenestra has a sister relationship with another putative new species, Heterodera sp. 2 from Mexico, which is not yet morphologically characterised.
Fig. 4. Scanning electron microscopy (SEM) of the terminal pattern and cone of a young cyst of Heterodera sp. 5 from Tanzania. A: Frontal view showing the vulval opening (arrow); B: Ventral view showing the anal opening (arrow).
Phylogeny of Heterodera from monocots
Presently, the Cardiolata group or H. cardiolata species complex (Subbotin et al., 2010b) consists of H. cardiolata, H. graminis Stynes, 1971, H. longicolla Golden & Dickerson, 1973, H. phragmitidis Kazachenko, 1986 and an undescribed Heterodera sp. 4 found in the Ukraine and Iran. Heterodera sp. 4 was briefly characterised by Subbotin et al. (2000) and Tanha Maafi et al. (2003). Based on the comparison of morphological and morphometric characters of H. cardiolata and H. cynodontis Shahina & Maqbool, 1989, Subbotin et al. (2010b) proposed to consider H. cynodontis as a synonym of H. cardiolata and retain H. graminis from Australia as a valid species until detailed molecular analysis of all species from the H. cardiolata species complex are made. In this study, the COI sequences of the putative new species Heterodera sp. 4 were supplied for the first time.
Heterodera sp. 1 (Sacchari group) and Heterodera sp. 5 (Afenestrata group) (Fig. 4) were collected in one location in Tanzania. Heterodera sp. 5 was preliminary identified as H. africana (Luc, Germani & Netscher, 1973) Mundo-Ocampo et al., 2008; however, additional studies require confirming this identification. To the best of our knowledge, there are no reports of cyst nematodes from Tanzania. Subbotin et al. (2010a) listed 18 species of the genus Heterodera from Africa, mainly in North and West Africa. Three species: H. africana (Afenestrata group), H. gambiensis Merny & Netscher, 1976 (Sacchari group), H. sacchari (Sacchari group) were reported only from Africa and were not found in other continents.
The study confirmed the conclusions (Subbotin et al., 2018) that each cyst nematode species has a unique COI sequence set or DNA barcode that enables its identification and separation from all other species. DNA barcoding can be considered as a powerful tool for identification of cyst nematodes species. DNA sequences of this gene might serve as an important information basis for developing specific diagnostics tools, including PCR-RFLP, conventional PCR and Real Time PCR with species-specific primers and probes, as well as other techniques.
ACKNOWLEDGEMENTS
The authors thank Drs D. Hunt and M. Mundo-Ocampo for providing cyst nematode samples. This work was sponsored by USDA APHIS Farm Bill grant: AP18PPQS&T00C201 (agreement no. 18-0430-000-FR). The reported study on African cyst nematode species was partly funded by RFBR and NRF according to the research project no. 19-51660001.
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S.A. Subbotin, A. Swart, I. Cid del Prado Vera, Z. Tanha Maafi and V.N. Chizhov. Штрих-кодирование ДНК и филогения некоторых видов цистообразующих нематод из рода Heterodera (Tylenchida: Heteroderidae), паразитирующих на однодольных.
Резюме. В настоящее время род Heterodera включает 87 валидных видов, из которых пять видов: H. bifenestra, H. cardiolata, H. cyperi, H. goldeni и H. sacchari охарактеризованы в данном исследовании на молекулярном уровне. Используя молекулярные критерии, мы также выделили пять предполагаемых новых видов: Heterodera sp. 1-sp. 5 из групп Afenestrata, Cardiolata, Bifenestra и Sacchari. Всего было получено 16 новых последовательностей ITS рРНК гена, четыре новых последовательности D2-D3 сегмента 28S рРНК гена и 23 новые частичные последовательности COI гена из 14 популяций цистообразующих нематод, собранных в девяти странах: Ганы, Ирана, Мексики, Пакистана, России, Испании, Танзании, Нидерландов и Украины. Филогенетические отношения внутри рода Heterodera проанализированы и представлены с использованием этих трех фрагментов генов. Исследование подтвердило вывод о том, что каждый вид цистообразующих нематод имеет уникальную последовательность COI гена или штрих-код ДНК, который позволяет идентифицировать виды друг от друга.
