Russian Journal of Nematology, 2020, 28 (1), 1 – 28
Molecular characterisation of some plant-parasitic nematodes (Nematoda: Tylenchida) from Belgium Catherine Malike Etongwe1, Phougeishangbam Rolish Singh1, Wim Bert1 and Sergei A. Subbotin2, 3 1Nematology Research Unit, Department of Biology, Ghent University, K.L. Ledeganckstraat 35, 9000, Ghent, Belgium 2Plant Pest Diagnostic Centre, California Department of Food and Agriculture, 3294 Meadowview Road, 95832-1448, Sacramento, CA, USA 3A.N. Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences, Leninskii Prospect 33, 117071, Moscow, Russia e-mail: [email protected]
Accepted for publication 20 February 2020
Summary. Using morphological, morphometric and molecular analysis, eleven valid nematode species from nine genera: Amplimerlinius icarus, Criconema annuliferum, Criconemoides informis, Helicotylenchus varicaudatus, Hemicriconemoides pseudobrachyurus, Hemicycliophora thienemanni, Mesocriconema xenoplax, Paratylenchus bukowinensis, P. nanus, Rotylenchus montanus and R. robustus together with twelve unidentified species, were identified in samples collected from eighteen locations in Belgium. The unidentified species include six Paratylenchus species, one Helicotylenchus species, three criconematid species and two Rotylenchus species. A total of new partial 21 18S rRNA, 69 28S rRNA, 10 ITS rRNA and 51 COI mtDNA gene sequences were obtained and used for phylogenetic and sequence analysis. Short descriptions, morphometrics and light and scanning microscopic photos are presented for selected species. Based on the results of molecular analysis, Hemicriconemoides promissus syn. n. was proposed as a junior synonym of H. pseudobrachyurus. Key words: Amplimerlinius icarus, Criconema annuliferum, Criconemoides informis, Helicotylenchus varicaudatus, Hemicriconemoides pseudobrachyurus, Hemicycliophora thienemanni, Mesocriconema xenoplax, Paratylenchus bukowinensis, Paratylenchus nanus, Rotylenchus montanus, Rotylenchus robustus, phylogeny, 18S rRNA, ITS rRNA, 28S rRNA, COI mtDNA.
The rapid identification of potentially harmful even distorted species (Eyualem & Blaxter, 2003; plant-parasitic nematodes is of critical importance in Bhadury et al., 2006; Miller, 2007; Ferri et al., the design of effective management strategies and 2009; Yao et al., 2010; Ahmed et al., 2016; control measures (Gonçalves de Oliveira et al., Subbotin et al., 2018). At the same time, 2011). Despite the immense risk potential, the classification, species delineation, revision and morphological diagnosis of plant-parasitic understanding of phylogeny currently depends nematodes is often rendered a difficult task due to greatly on the usage of a suitable DNA domain their high phenotypic plasticity and absence of clear within a nuclear or mitochondrial genome (Bae et diagnostic characters (Vovlas et al., 2008; al., 2009; Van den Berg et al., 2013). Due to the Palomares-Rius et al., 2017). decreasing price and increased availability of Molecular taxonomy and DNA barcoding sequencing instruments, the number of nematode provide powerful tools for the identification of species that have been sequenced has grown organisms, especially those organisms for which exponentially the last decades. However, despite morphological diagnostic characteristics are scarce this growth in work rate, the vast majority of (Hebert et al., 2003). Their use is particularly morphospecies remain unlinked to DNA sequences. advantageous because of the rapidity, accuracy and Moreover, a substantial part of the existing sequence transferability of the methods, allied with their data appears to be incorrect, with faults ranging ability to be used in identification irrespective of life from sequencing errors over misassembles to stage of the organism. Furthermore, they enable the misidentified, mislabelled, or chimera sequences. discrimination of cryptic, very closely-related and Therefore, to design a reliable diagnostic strategy, it
1 © Russian Society of Nematologists, 2020; doi: 10.24411/0869-6918-2020-10001 C.M. Etongwe et al. is of utmost importance to safeguard the link (Amiri et al., 2003), Meloidogyne artiellia (Damme between morphospecies and species-specific et al., 2013), Paratrophurus bursifer (Consoli et al., sequences, i.e. molecular barcodes (Janssen et al., 2017), Pratylenchus brzeskii (Janssen et al., 2017a), 2017a; Qing et al., 2020). Pratylenchus convallariae (Janssen et al., 2017b), Belgium has a long-standing tradition of Abursanema quadrilineatum (Qing et al., 2017), conducting diverse nematode-based studies, Malenchus cylindricus (Qing et al., 2018), resulting in a relatively well-studied nematofauna. Pratylenchus horti (Nguyen et al., 2019a), Coomans (1989) provided a thorough list of Belgian Rotylenchus buxophilus (Nguyen et al., 2019c), nematofauna, albeit excluding animal-parasitic Scutellonema brachyurus and Meloidogyne nematodes and Bert et al. (2003) listed the incognita (Nguyen et al., 2019b). Tylenchomorpha (Tylenchida and Aphelenchida) of The goals of the present study were: i) to carry Belgium. More recently, Steel et al. (2014) have out morphological and morphometric provided an updated list of Belgian nematofauna characterisation of species from the Criconematidae, comprising 418 species, of which 170 are Hoplolaimidae, Hemicycliophoridae, Dolicho- Tylenchida. At the time of writing this paper, the doridae and Paratylenchidae families collected in authors are aware of 13 tylenchid species that Belgium; and ii) to provide molecular should be added to the current Belgian nematofauna characterisation of selected species using the D2-D3 list: Heterodera ustinovi (Subbotin et al., 2000), of the 28S rRNA, ITS rRNA, 18S rRNA and COI H. aucklandica (Subbotin et al., 2003); H. betae mtDNA gene sequences.
Table 1. Nematode species and populations used in this study.
Sample Locality GPS location Species code Ghent University Botanical BE1 51°2’7.53” N; 3°43’20.07” E Helicotylenchus sp. A, Paratylenchus nanus Garden Ghent University Botanical BE2 51°2’7.53” N; 3°43’20.07” E Paratylenchus sp. C, Rotylenchus robustus Garden Ghent University Botanical BE4 51°2’7.10” N; 3°43’19.28” E Paratylenchus sp. B, R. robustus Garden Ghent University Botanical BE5 51°2’7.55” N; 3°43’20.47” E Helicotylenchus sp. A Garden BE7 Blaarmeersen (Ghent) 51°02’39” N; 3°41’08” E Helicotylenchus varicaudatus
BE9 Ghent, Citadel Park 51°02’05” N; 3°43’10” E Paratylenchus sp. E, H. varicaudatus, Mesocriconema xenoplax Hemicriconemoides pseudobrachyurus, P. nanus, R. robustus, BE11 Zwijnaarde 51°00’19” N; 3°42’11” E H. varicaudatus BE13 Zwijnaarde 51°00’17” N; 3°42’10” E Mesocriconema xenoplax, R. robustus, Criconema annuliferum
BE14 De Panne 51°07’14” N; 2°39’29” E Criconemoides informis, Helicotylenchus sp. A, H. varicaudatus C. annuliferum, criconematid sp. B, Rotylenchus sp. B, BE15 Kortrijk 50°47’58” N; 3°11’37” E Amplimerlinius icarus BE16 Kortrijk 50°48’29.81” N; 3°12’30.79” E C. annuliferum
BE18 Blaarmeersen 51°07’14” N; 2°39’29” E C. informis, Paratylenchus sp. F Hemicycliophora thienemanni, criconematid sp. B, BE19 Blaarmeersen 51°02’18.9” N; 3°41’17.2” E Paratylenchus bukowinensis, Paratylenchus sp. 8, Paratylenchus sp. B, Paratylenchus sp. F Mesocriconema xenoplax, Paratylenchus sp. D, BE20 Blaarmeersen 51°02’14” N; 3°41’23” E Paratylenchus sp. B, criconematid sp. A, criconematid sp. C, C. annuliferum, Rotylenchus sp. A, H. varicaudatus BE21 Merendree 51°4’30.49” N; 3°34’39.65” E M. xenoplax, C. annuliferum
BE22 Merendree 51°04’12” N; 3°34’37” E M. xenoplax, C. informis, Paratylenchus sp. F
BE23 Heusden, Destelbergen 51°01’24.01” N; 3°80’69.86” E Rotylenchus montanus
BE24 Eine, Oudenaarde 50°87’73.07” N; 3°58’17.62” E A. icarus
2 Molecular characterisation of plant-parasitiic nematodes from Belgium
Fig. 1. Hemicriconemoides pseudobrachyurus female from Belgium. A & B: Scanning ellectron microscopic (SEM) photos of anterior region; C & D: SEM photos of posterior region; E: Light microscopicc (LM) photos of posterior region; F & G: LM photos of anterior region. F and G have same scale.
Light microscopy. Nematodes used for light MATERIAL AND METHODS microscopy were killed by gentle heat, fixed in Trump’s fixative [2% parraformaldehyde + 2.5% Nematode populations sampling and glutaraldehyde in a 0.1 M Sorenson buffer (sodium extraction. Soil samples were collected using an phosphate bufffer at pH 7.3)], transferred to auger at a depth of 15-30 cm from several locations anhydrous glycerin (De Griisse, 1969) and mounted in Belgium (Table 1). Samples were randomly on permanent slides followiing the method of Singh collected from the rhizosphere of several et al. (2018). Photographs aand measurements were unidentified plants and trees with different soil made from both temporal and permanent slides by characteristics, vegetation and topology. using an Olympus BX5 DIIC Microscope equipped Approximately 500 g of soil was put in pllastic with an Olympus C5060Wz camera. The containers, labelled and kept at 4°C. Nemattodes measurements were made using ImageJ 1.51 under were extracted from soil at the Nematology 10×, 20×, 40× and 100× magnifications. Holotype Research Unit of Ghent University, using the slide R 766 and paratypes sllides (R 324-665-715) of modified Baermann tray technique (Whitehead & Hemicriconemoides pseudobrachyurus obtained Hemming, 1965), Cobb’s sieving and decanting from the Department of Plants and Crops, Faculty of method using two sets of sieves (1000 µm mesh to Bioscience Engineering, Ghent University were remove debris, then 38 µm mesh for the collection used in this study for comparison with of the nematodes) followed by sugar centrifugation Hemicriconemoides species collected during this method (Jenkins, 1964). study.
3 C.M. Etongwe et al.
Fig. 2. The 50% majority rule consensus tree obbtained using Bayesian analysis of the 18S rRNA gene sequence alignment with the GTR+I+G model. Posterior probability of over 70% is given as a percentage for each appropriate clade. New sequences are indicated in bold. * – identified as Paratylenchus nanus by van Megen et al. (unpublished); ** – identified as Hemicriconemoides sp. by Pham & Zheng (unpublished); *** – identified as Rotylenchus uniformis by Helder et al. in the GenBank; **** – identified as Rotylenchus robustus by van Megen et al. (2009).
4 Molecular characterisation of plant-parasitiic nematodes from Belgium
Fig. 3. The 50% majority rule consensus tree obbtained using Bayesian analysis of the 28S rRNA gene sequence alignment with the GTR+I+G model. Posterior probability of over 70% is given as a percentage for each appropriate clade. New sequences are indicated in bold. * – identified as Helicotylenchus sp. IX-4 by Subbotin et al. (2015); ** – identified as Rotylenchus uniformis by Subbotin et al. (2007); *** – identified as Rotylenchus sp. by Subbotin et al. (2007); **** – identified as Helicotylenchus sp. IX-3 by Subbotin et al. (2015).
5 C.M. Etongwe et al. Scanning microscopy. For scanning electron µl dNTP (10 mM); 0.5 µl of each primer; 0.05 µl microscopy (SEM), specimens fixed in Trump’s Toptaq) and 2 µl of DNA template. The fixative were washed in 0.1 M phosphate buffer (pH thermocycling profile for 18S rRNA gene consisted = 7.5) and dehydrated in a graded series of ethanol of 5 min at 94°C, 35 cycles of 1 min at 94°C, 1 min solutions, critical-point-dried with liquid CO2, 30 s at 52°C, and 2 min at 68°C, followed by a final mounted on stubs with carbon tabs (double step of 10 min at 68°C. The PCR amplification conductive tapes), coated with gold of 25 nm, and profile for ITS and 28S rRNA genes consisted of photographed with a JSM-840 EM (JEOL) at 12 kV 4 min at 94°C, 35 cycles of 1 min at 94°C, 1 min (Singh et al., 2018). 30 s at 55°C, and 2 min at 72°C, followed by a final DNA extraction. Live nematode specimens were step of 10 min at 72°C. handpicked using a picking needle under a binocular The forward primer JB3 (5’-TTT TTT GGG microscope, put into a drop of water on a glass slide, CAT CCT GAG GTT TAT-3’) and the reverse killed by gentle heat and temporary slides sealed with primer JB4 (5’-TAA AGA AAG AAC ATA ATG paraffin wax. Pictures and measurements were made AAA ATG-3’) as described by Derycke et al. using the instruments mentioned above. This was (2010) were used for amplification of the COI gene done to maintain a link between morphology and for the majority of nematodes, whereas the forward molecular data of species obtained. Later the primer COIF5 (5’-AAT WTW GGT GTT GGA nematodes were removed from the temporary slides, ACT TCT TG AAC-3’) and the reverse primer picked and put in 20 μl double distilled water on a COIR9 (5’-CTT AAA ACA TAA TGR AAA TGW glass slide, cut into two or more pieces using a GC W ACW ACA TAA TAA GTA TC-3’) as stainless-steel dissecting blade under a dissecting described by Powers et al. (2014) were used for microscope. The cut nematodes were transferred into amplification of the COI gene from criconematids. a 0.2 ml Eppendorf tube, containing 20 μl worm lysis For the partial COI gene study, 3 μl of extracted buffer (50 mM KCl; 10 mM Tris pH 8.3; 2.5 mM DNA was added to a 0.2 ml Eppendorf tube MgCl2; 0.45% NP 40 (Tergitol Sigma); 0.45% Tween containing 23 µl of Mastermix as described above. 20) for DNA extraction. The tubes were kept at For specimens from the Criconematidae family, −20°C for 10 min after which 1 μl proteinase K (1.2 10 μl DreamTaq Green PCR Master Mix (2×) mg ml−1) was added into the Eppendorf tubes and (Thermo Fisher Scientific), 10 μl water and 0.15 μl tubes were incubated in a PCR machine at 65°C (1 h) of each primer (1.0 μg μl–1) was used. The PCR and 95°C (10 min) consecutively. After incubation, amplification profile for the JB3/JB4 primers the tubes were centrifuged and kept at −20°C until consisted of 3 min at 94°C, 34 cycles of 30 s at use in the DNA database of the Nematology 94°C, 30 s at 45°C, and 1 min at 72°C, followed by Research Unit of Ghent University, Belgium. a final step of 10 min at 72°C. The PCR PCR and DNA sequencing. Two µl DNA amplification profile for the COIF5/COIR9 primers template was used from the tubes for PCR. Four consisted of 4 min at 94°C, followed by 40 cycles of gene fragments were amplified and sequenced for 1 min at 94°C, 1 min at 45°C, and 1 min 30 s at this study: 18S rRNA, the ITS rRNA, the D2-D3 72°C, with a final extension at 72°C for 10 min. expansion segments of 28S rRNA and the partial Five μl of the PCR product was run on a 1% TAE- COI genes. The forward primer SSU18A (5’-AAA buffered agarose gel (130 V, 20 min). PCR products GAT TAA GCC ATG CATG-3’) and the reverse were purified using QIAquick PCR Purification Kit primer SSU 26R (5’-CAT TCT TGG CAA ATG (Qiagen) and directly sequenced with the primers CTT TCG-3’) as described by Mayer et al. (2007) mentioned above by Macrogen Inc. The new were used for amplification of 18S rRNA gene, For sequences were submitted to the GenBank database the ITS rRNA the forward primer TW81 (5’-GTT under the accession numbers: MN783679- TCC GTA GGT GAA CCT GC-3’), and the reverse MN783742, MN783658-MN783678 as indicated in primer AB28 (5’-ATA TGC TTA AGT TCA GCG phylogenetic trees (Figs 2-4). GGT-3’) as described by Curran et al. (1994) were Phylogenetic and sequence analysis. used. The forward primer D2A (5’-ACA AGT ACC Alignments with the 18S rRNA, ITS rRNA, 28S GTG AGG GAA AGT TG-3’), and reverse primer rRNA and COI gene sequences were created using D3B (5’-TCG GAA GGA ACC AGC TAC TA-3’) Clustal X 1.83 (Thompson et al., 1997) with default as described by Subbotin et al. (2006) were used for parameters. New sequences were aligned with amplification of the D2-D3 expansion segments of corresponding gene sequences available in the 28S rRNA gene. For amplification of rRNA genes, GenBank (Zeng & Ye, unpublished; van Megen et the PCR reaction included Mastermix (17 µl ddH2O; al., unpublished; Holterman et al., 2006, 2009, 2.5 µl 10× buffer; 2 µl MgCl2; 2.5 µl Coralload; 0.5 unpublished; Ghaderi et al., unpublished; Powers et
6 Molecular characterisation of plant-parasitic nematodes from Belgium al., 2010, 2014, 2016a, b, 2017; Munawar et al., obtained: 21 – partial 18S rRNA gene, 69 – D2-D3 of unpublished; Subbotin et al., 2005, 2006, 2007, 28S rRNA gene, 10 – ITS rRNA gene and 51 – COI 2014, 2015; Bert et al., 2008; Vovlas et al., 2008; mtDNA gene. Species delimitation was performed Van den Berg et al., 2012, 2013, 2014a, b, 2015, using an integrated approach that considered 2017, 2018; Cantalapiedra-Navarrete et al., 2013; morphological and morphometric evaluation Ghaderi et al., 2014; Panahandeh et al., 2014; combined with molecular criteria based on Esmaeili et al., 2016; Olson et al., 2017; Maria et phylogenetic inference and sequence analyses. al., 2018, 2019; Rybarczyk-Mydlowska et al., 2019, Eleven valid species from nine genera were identified unpublished; Tandingan De Ley et al., unpublished; during this study namely: Amplimerlinius icarus, Wang et al., unpublished; Holroyd et al., Criconema annuliferum, Criconemoides informis, unpublished; Cordero et al., 2012; Kanzaki & Futai, Helicotylenchus varicaudatus, Hemicriconemoides 2002; Pang et al., unpublished; Golhasan et al., pseudobrachyurus, Hemicycliophora thienemanni, 2016; Alvani et al., unpublished; Helder et al., Mesocriconema xenoplax, Paratylenchus unpublished; Meldal et al., 2007; Pham & Zheng, bukowinensis, P. nanus, Rotylenchus montanus and unpublished; Ortiz et al., 2016; Jin, unpublished; R. robustus. Twelve putative species of several Afshar et al., 2019; Tzortzakakis et al., 2016; genera were also identified: six – Paratylenchus Noruzi et al., 2015; Skwiercz et al., unpublished; (Paratylenchus sp. B, Paratylenchus sp. C, Schratzberger et al., unpublished; Azizi et al., 2016, Paratylenchus sp. E, Paratylenchus sp. 8, unpublished; Karegar et al., unpublished; Yang, Paratylenchus sp. D and Paratylenchus sp. F), one unpublished; Alvani et al., unpublished; Tabolin & Helicotylenchus (Helicotylenchus sp. A), three Markina, unpublished; Pang et al., unpublished; criconematids (criconematid sp. A, criconematid sp. Nguyen et al., 2019c). B, criconematid sp. C), and two Rotylenchus The sequence alignments were analysed with (Rotylenchus sp. A, Rotylenchus sp. B). Bayesian inference (BI) using MrBayes 3.1.2 Identifications of these species at species level have (Ronquist & Huelsenbeck, 2003). The best-fit model not been done because of the limited number of of DNA evolution was obtained using the program specimens. jModelTest 0.1.1 (Posada, 2008) with the Akaike Samples contained from one up to eight Information Criterion. The BI analysis for each gene nematode species of the different studied genera. was initiated with a random starting tree and was run The most diverse sample was Blaarmeersen (BE20), with four chains for 1.0 × 106 generations. The which had representative species from six genera. Markov chains were sampled at intervals of 100 Most samples consisted of a single species per generations. Two runs were performed for each genus (monospecific), except for some (BE19 and analysis. After discarding burn-in samples and BE20) that contained more than one species of evaluating convergence, the remaining samples were Paratylenchus. retained for further analysis. The topologies were MORPHOLOGICAL AND MOLECULAR used to generate a 50% majority-rule consensus tree. CHARACTERISATION Posterior probabilities (PP) were given on appropriate Hemicriconemoides pseudobrachyurus clades. Trees were visualised with the TreeView 1.6 De Grisse, 1964 (Fig. 1) program and drawn with Adobe Illustrator v.10. Pairwise divergence between taxa was Hemicriconemoides pseudobrachyurus was first calculated as the absolute distance value and the described from sandy soil around roots of grass percent of mean distance, with adjustment for plants and potatoes in Huise (Kruise), Belgium (De missing data, using PAUP* 4b10 (Swofford, 2003). Grisse, 1964). During our study, the attempt to The alignment for ITS rRNA sequences was collect H. pseudobrachyurus from the type locality used to construct phylogenetic network estimation failed; however, several female specimens of this using statistical parsimony (SP) as implemented in species were collected from a location in the POPART software (http://popart.otago.ac.nz) proximity from the type locality. Morphological and (Bandelt et al., 1999). morphometric characters of this H. pseudobrachyurus population collected from a RESULTS AND DISCUSSION geographically close (15 km away) and similar habitat (Eedstraat, Zwijnaarde) agreed with the Species identification and delimitation. During original description. This population is compared this study, 173 nematodes were taken for with the populations of H. promissus collected from morphological and molecular study from 18 locations different localities by other authors (Table 2). in Belgium. A total of 121 new sequences was Measurements are given in Table 2.
7 C.M. Etongwe et al.
et al.
(2014b)
Bolonia, Spain an ± s.d. (range). Van den Berg
et al.
syn. n.)
Spain
(2014b)
Van den Berg Monteagudo Island, H. promissus m and in the form: me form: the in and m (=
μ
(2006) (2006) 0.8-25.5) 0.8-25.5) (20.0-28.6) 25.7 ± 3.0 (20.8-25.4) 22.8 ± 2.1
(11.2-13) (11.8-15.8) 13.9 ± 1.2 (11.2-13) 12.2 ± 0.8 et al.
Spain 0.9 (7.4-10.2) (7.4-10.2) 0.9 (10.4-13.6) 11.9 ± 1.1 (8.8-10.7) 9.8 ± 0.8 10 females 10 females 16 females Bolonia (Cádiz), Bolonia (Cádiz),
Vovlas H. pseudobrachyurus
. All measurements are in
Torre Canne, Canne, Torre Vovlas (1980) Brindisi, Italy
Huise, Belgium Huise, Belgium De Grisse (1964)
Hemicriconemoides pseudobrachyurus
H. pseudobrachyurus
1 1 1 (1-2) 1 ± 0.5 1 1 Belgium Belgium
10 females 20 females 20 females Zwijnaarde, Zwijnaarde, 2.8 ± 0.5 (2-3) (2-3) 2.8 ± 0.5 – – (5-6) 5.8 ± 0.5 – – (4-5) 4.4 ± 0.4 (4-5) 4.4 ± 0.5 6 to 7 (5-9) 6.3 ± 1.1 (1-2) 1.3 ± 0.5 Present study study Present 52 ± 3.5 (48-57) (48-57) 52 ± 3.5 (46-53) 50 ± 5.3 47 (45-49) 50 (43-54) – (45-54) 51 ± 3.2 (93-95) 95 ± 2.4 (55-60) 57 ± 1.6 95 (93-96) 55 (52-59) 94-95 49 (48-51) (94-96) 95 ± 1.0 (47-49) 48 ± 0.8 (92-94) 93.2 ± 0.8 (51-55) 52.9 ± 1.4 (94-96) 95.0 ± 1.0 (47-49) 48.0 ± 0.8 0.5 ± 0.1 (0.5-0.6) (0.5-0.6) 0.5 ± 0.1 0.5 (0.4-0.6) 1.7 (1.5-1.7) – – – 4.8 ± 0.5 (4.3-5.9) (4.3-5.9) 4.8 ± 0.5 (0.9-1.0) 0.9 ± 0.1 4.9 (4.2-5.7) (4.8-5.2) 4.9 ± 0.9 – 5.8 (5.5-6.4) – (46-48) 47.6 ± 2.0 (4.1-5.3) 4.6 ± 0.5 (3.9-4.7) 4.2 ± 0.4 (4.0-5.4) 4.6 ± 0.5 (4.1-5.3) 4.6 ± 0.5 – (38-45) 41.4 ± 2.6 – (5.1-6.3) 5.9 ± 0.4 – – (15-25) 22.2 ± 3.1 – (12-16) 14.0 ± 1.4 – (18-23) 21.6 ± 1.6 – (27-30) 28.9 ± 1.0 – – 11 (9-13) – – (0.9-1.4) 1.1 ± 0.2 25 (23-29) – (0.7-1.0) 0.8 ± 0.1 – (7.3-9.2) 8.3 ± 0.8 (0.95-1.05) 0.04 ± 0.99 8 to 9 – – 24 (23-26) – (8.3-10.4) 9.1 ± 0.9 – 15 (14-16) (23-26) 25 ± 1.3 (8-10) 9 ± 1.0 (43-47) 44.7 ± 1.8 – 5-6 0.9 (0.8-1.0) (20-26) 22.3 ± 1.9 (15-17) 16 ± 1.0 (36-38) (11-14) 37.3 ± 1.0 12.2 ± 0.9 (2.5-3.0) 2.9 ± 0.3 – (23-26) 24.5 ± 1.3 (19-25) 20.9 ± 1.8 (0.9-1.0) 0.9 ± 0.1 (8-10) 8.8 ± 0.9 (28-35) 32.2 ± 2.4 (4.0-4.5) 4.3 ± 0.3 (15-17) 15.8 ± 1.0 (0.9-1.2) 1.1 ± 0.1 – (39-43) 40.3 ± 1.9 (14-21) 17.6 ± 2.2 (0.9-1.0) 0.9 ± 0.05 (5-6) 5.5 ± 0.6 (21-22) 21.5 ± 0.6 101 ± 4.2 (95-108) (95-108) 101 ± 4.2 98 (91-103) 97 (89-102) (92-100) 96 ± 4.1 (97-117) 105 ± 5.2 (92-100) 95.5 ± 4.1 536 ± 50 (452-591) 536 ± 50 (452-591) 531 (440-580) 580 (460-750) 491 ± 51 (438-700) 447 ± 43 (400-527) 491 ± 51 (438-560) (8.7-11.5) 9.6 ± 0.8 – 8.5-9.0 – (9.0-9.5) 9.3 ± 0.3 (8-9) 8.5 ± 0.6 258 ± 7.3 (250-268) (250-268) 258 ± 7.3 – (100-122) 111 ± 6.8 – 110 (97-128) – – (162-260) 225 ± 35.5 (242-324) 274 ± 35.5 89-101 (89-102) 97 ± 6.7 (104-110) 106 ± 2.6 10.7 ± 1.2 (9.7-12.8) (9.7-12.8) 10.7 ± 1.2 10.3 (9.2-11.8) 8.4 (6.8-10.4) 8.8 ± 148 ± 14.2 (126-166) (126-166) 148 ± 14.2 – 159 (155-165) – (90-104) 98 ± 6.1 (124-145) 130 ± 10.0 (11.3-13.3) 12.9 ± 0.8 (18.4-36.0) 24.7 ± 5.3 14 (12-17) 24 (17-31) (45.1-55.1) 49.1 ± 5.2 16 (15-20) 30 (27-35) 12.2 ± 0.8 (81.7-86.3) 83.6 ± 1.6 – (2 22.8 ± 2.1 – – – – – (40.5-58.2) 50.8 ± 5.9 (54.5-57.9) 55.8 ± 1.4 (81.7-85.5) 83.9 ± 1.6 (75-80.8) 77.6 ± 2.4
Species
Morphometric comparison of
(2014) and Geraert Characters* (abbreviations Characters* (abbreviations are Subbotin as defined by et.al. (2010)) N VL/ST pharynx St% L a b c c’ o DGO V G1 Ovary length Stylet length length Metenchium M Stylet knob height Styletwidth knob pore excretory to end Anterior body Diam at mid Annulus width Tail length Pharynx length R RSt Roes Rex Rvan Ran VL/VB St%L Table 2.
8 Molecular characterisation of plant-parasitic nematodes from Belgium Description. Female. Body cylindrical and that H. promissus can be further separated from slightly curved ventrally when heat-killed. Closely H. pseudobrachyurus by its slightly shorter pharynx fitted sheath with smooth annuli, attached at the (89-101 vs 97-128 μm) extending for 15-17 vs 16-23 anterior end and the vulva. No lateral line present. body annuli; presence of spermatheca filled with Anastomosis of annuli not seen. Cephalic region spermatozoids in all females in both, Italian and truncated with two sublateral legs and body annulus. Spanish populations vs presence of spermatheca but Cephalic region collapsed after fixation. Stylet long never clearly visible spermatozoids in and robust with strong anchor-shaped knobs H. pseudobrachyurus. The male was not known for anteriorly indented and rounded posteriorly. Dorsal this species. Geraert (2010) did also not support this pharyngeal gland opening situated very close to the synonymisation and listed H. promissus as a valid stylet base. Excretory pore situated 7-9 annuli species. Our study also showed similar differences posterior to base of the pharyngo-intestine junction. between H. pseudobrachyurus and H. promissus; Hemizonid not seen. Vulva located usually on the however, because there are high similarities in the sixth (6-7) annulus from the tail terminus, open and 18S rRNA (0.4%), D2-D3 of 28S rRNA (0.7%), ITS without vulva flap. Vagina straight, oblique rRNA (0.5%) and COI (4.6%) gene sequences of anteriorly directed. Spermatheca relatively large, both species, we consider these species to be co- round, filled with rounded spermatozoids. Anus one specific and their morphological differences is a annulus away from the vulva. Tail conical ending result of geographical variation. We suggest that with a small finely rounded lobe (Voucher slides H. promissus syn. n. should be considered as a UGnem 240-241; Nematology Research Unit, Ghent junior synonym of H. pseudobrachyurus. University). Mesocriconema xenoplax Male and juvenile. Not found. (Raski, 1952) Loof, 1989 Molecular characterisation. Intraspecific sequence variation for H. pseudobrachyurus was 0- This nematode has a worldwide distribution and 0.8% (0-7 bp), 0-0.7% (0-4 bp) and 2.1-4.6% (8-17 wide host range (Karanastasi et al., 2008). It is bp) for 18S rRNA, D2-D3 of 28S rRNA and COI known for damaging the roots of plants. In Belgium, genes, respectively. Phylogenetic positions of this it was found parasitising turf grass (Vandenbossche species within some tylenchids are given in Figures et al., 2011). In this study, two populations of this 2, 3 and 4. The ITS alignment for species from different locations (Zwijnaarde and H. pseudobrachyurus included five sequences and Merendree) have been morphologically and was 803 bp in a length. Three new ITS sequences molecularly characterised. were obtained during this study. Maximal sequence Measurements are given in Table 3. diversity was 0.7% (5 bp). The ITS haplotype Description. Female. Body ventrally curved to phylogenetic network is given in Figure 5A. C-shaped when fixed. Cuticular annuli retrorse, with Synonymisation of H. promissus Vovlas, 1980 faint longitudinal lines seen. Anastomoses seen in syn. n. with H. pseudobrachyurus. Morphology all specimens observed at the cephalic region, mid- and morphometrics of Hemicriconemoides body and vulva. Cephalic region continuous with specimens collected in this study agreed with the body. Labial disc elevated with two sub-median original description and type material of lobes seen projecting anteriorly. Stylet robust with H. pseudobrachyurus. This species is very similar strong knobs. In one specimen the reproductive with H. promissus described by Vovlas (1980) from system was reflexed. Spermatheca rounded, vagina the rhizosphere of halophilic plants from a sandy sigmoid. Tail bilobed. dune of Torre Canne, Brindisi, Italy. Male. Not found. Hemicriconemoides promissus was considered to be Molecular characterisation. Intraspecific morphologically very similar to sequence variation for M. xenoplax was 0-0.4% (0-5 H. pseudobrachyurus, but was considered different bp), and 0-0.5% (0-2 bp) for D2-D3 of 28S rRNA based on the length of the stylet (48-51 vs 52-59 and COI genes, respectively. Phylogenetic positions µm), RSt (8-9 vs 9-13) and the higher value of the of this species within some tylenchids are given in ratio of vulva terminus distance divided by the stylet Figures 3 and 4. The ITS rRNA gene alignment for length (VL/ST = 1.5-1.7 vs 0.39-0.62). Germani & M. xenoplax included 30 sequences, including four Anderson (1991) synonymised H. promissus with new sequences obtained during this study. The H. brachyurus but Vovlas et al. (2006) re- alignment was 315 bp in the length. Maximum established H. promissus as valid species and intraspecific ITS sequence variation was 1.7% (0-5 differentiated it from H. brachyurus and bp). The ITS haplotype phylogenetic network is H. pseudobrachyurus. Vovlas et al. (2006) indicated given in Figure 5B.
9 C.M. Etongwe et al.
153 – Criconemoides informis Criconemoides
from Begium.
101.3) 67.2 61.5
0 ± 1.0 (11.0-13.5) (11.0-13.5) 0 ± 1.0 10.7 11.3 (11.0-26.5) 7 ± 6.2 10.7 10.7 80.5 ± 7.9 (66.5-85.4) (66.5-85.4) 80.5 ± 7.9 53.0 46.5 26.1 ± 4.7 (19.4-30.9) (19.4-30.9) 26.1 ± 4.7 23.3 32.8 179 ± 13.0 (170-193.0)
63.3 ± 3.5(60.0-67.7) – 51.4 Criconemoides informis Criconemoides
and
Criconema annuliferum
2) 2) (47.3-59.5) 53.9 ± 4.4 (52.5-65.3) 56.4 ± 5.2 49.3 57.2 12.9) 12.9) (11.7-12.6) 12.2 ± 0.4 12. 16.6) (11.7-14.4) 12.6 ± 1.1 15. -5.9) (4.1-6.3) 5.2 ± 1.0 (2.2-2.9) 2.6 ± 0.3 3.6 4.3 -1.1) -6.1) (0.7-1.0) 0.8 ± 0.1 (5.7-8.6) 6.8 ± 1.1 (0.7-1.0) 0.8 ± 0.1 (7.1-10.9) 9.5 ± 1.5 0.6 8.9 0.5 11.6 -4.4) (3.6-4.3) 3.9 ± 0.3 (4.0-4.3) 4.1 ± 0.1 4.3 3.7 -9.6) (8.1-10.5) 9.6 ± 1.0 (9.0-11.0) 10.0 ± 0.7 10.9 7.4 5-62.3) 5-62.3) (44.5-55.6) 49.5 ± 5.6 (49.6-64.4) 54.8 ±5.8 46.5 – 0-80.8) 0-80.8) (77.3-90.0) 86.0 ± 5.0 2-26.4) 2-26.4) (23.8-39.1) 30.7 ± 6.2
8.0-56.4) (46.8-70.7) 56.0 ± 9.4 males 7 females 5 females 1 female 1 female Criconema annuliferum
± s.d. (range). mean and in the form: m , μ
esocriconema xenoplax
are in All measurements M
Mesocriconema xenoplax
10 females 10 females 10 fe 95 ± 2.0 (94-96) (94-96) 95 ± 2.0 (93-95) 93 ± 0.6 (89-90) 89 ± 0.5 (89-90) 90 ± 0.6 (89-91) 90 ± 0.8 92 92 4.8 ± 0.8 (3.8-5.7) (3.8-5.7) 4.8 ± 0.8 (2.5-5.2) 3.5 ± 1.2 (4.1 5.3 ± 0.7 0.8 ± 0.2 (0.5-0.9) (0.5-0.9) 0.8 ± 0.2 (0.7-0.9) 0.8 ± 0.1 (0.7 0.9 ± 0.1 7.6 ± 0.8 (6.4-8.6) (6.4-8.6) 7.6 ± 0.8 (6.1-7.7) 6.9 ± 0.7 (3.4-6.0) 4.7 ± 1.1 (6.1-9.3) 7.0 ± 1.3 (7.2-10.9) 9.4 ± 1.5 6 7.1 (3.9-4.3) 4.1 ± 0.2 (3.5-4.6) 4.0 ± 0.5 (3.7 4.0 ± 0.2 9.9 ± 1.1 (8.4-11.5) (8.4-11.5) 9.9 ± 1.1 (10.2-14.5) 12.1 ± 1.8 (3.6 4.9 ± 1.0 170 ± 18.4 (150-191) (150-191) 170 ± 18.4 (118-142) 127 ± 13.4 (85.0-187) 133 ± 46.6 (155-181) 168 ± 10.6 574 ± 32.0 (526-611) (526-611) 574 ± 32.0 (392-693) 505 ± 115.4 (478-608) 542 ± 40.5 (482-578) 515 ± 42.6 (527-586) 564 ± 24.4 538 422 12.0 ± 0.8 (10.7-12.8) (10.7-12.8) 12.0 ± 0.8 (9.3-12.0) 10.7 ±1.4 (11.0- 11.7 ± 0.7 77.2 ± 1.9 (75.5-79.8) 80.6 ± 3.0 (75.3-82.7) 80.9 ± 2.5 (75.3-82.7) (55.9-131.0) 32.0 ± 85.5 81.4 (48.8-54.5) 50.9 ± 2.1 ± 6.8 (69.3-85.5) (35.6-50.0) 43.7 ± 6.0 (48.5-50.3) 49.7 ± 0.8 (35.4-43.9) 41.0 ± 4.6 78.7 61.6 (56.4-66. (45. 54.0 ± 7.2 75.6 76.3 ± 3.8 (73.1-82.6) 57.4 ± 3.9 (52.7-61.3) 95.0 ± 3.5 (90.3-99.5) (94.0-108.3) 5.9 ± 103.8 98.8±2.7 (95.8- 58.9 ± 4.3 (55.2-65.9) (55.2-65.9) 58.9 ± 4.3 (29.5-44.8) 14.1 ± 44.5 (68. 76.8 ± 4.6 12.5 ± 1.1 (11.1-13.9) (11.1-13.9) 12.5 ± 1.1 (10.5-18.1) 14.1 ± 3.1 (11.3- 12.9 ± 1.9 11.3 ± 0.7 (10.4-12.1) (10.4-12.1) 11.3 ± 0.7 (10.7-13.9) 11.7 ± 1.4 (8.2 8.7 ± 0.6 56.7 ± 11.1 (45.0-66.6) 43.7 ± 11.8 (27.6-55.2) 52.4 ± 4.1 (4 24.3 ± 15.8 (17.0-44.9) (17.0-44.9) 15.8 ± 24.3 (17.5-30.0) 22.0 ± 5.5 (18. 21.9 ± 2.9 of Morphometrics
Table 3. c o a b L V N c’ M G1 DGO length Species Species Width at
Merendree Blaarmeersen Merendree Zwijnaarde Kortrijk Characters Merendree Zwijnaarde Stylet length Metenchium Metenchium excretory pore pore excretory Excretory pore pore Excretory Telenchium legth Styletwidth knob from anterior end Stylet knob height Width at midbody
10 Molecular characterisation of plant-parasitic nematodes from Belgium
Begium. from
0 ± 4.1 (23.1-33.6) (23.1-33.6) 0 ± 4.1 23.1 (21.5-24.4) 4 ± 1.1 23.4 11.0 16.5 7 ± 2.4 (19.5-25.2) (19.5-25.2) 7 ± 2.4 19.1 (32.9-39.1) 6 ± 2.5 28.2 22.5 34.1 (16.4-18.2) 6 ± 0.8 12.5 14.6 Criconemoides informis 22.2 ± 4.4 (17.6-28.7) (17.6-28.7) 22.2 ± 4.4 13.6 12.8
and and
4) 4) (6.3-10.7) 8.5 ± 1.7 (8.5-10.2) 9.3 ± 0.7 7.0 9.4
annuliferum Criconema 30.1) 30.1) (18.3-23.0) 20.8 ± 1.7 27. 23.8) (20.4-22.4) 21.1 ± 0.9 22. 33.2) (26.9-30.7) 01.3 ± 28.8 30.8 ± 1.8 (28.6-33.4) 23.0 28.3 22.9) 22.9) (18.6-19.6) 19.1 ± 0.4 21. 38.2) (30.8-35.1) 32.9 ± 1.6 35. 20.2) (18.6-22.4) 20.3 ± 2.2 17. 139) 139) (125-141) 134 ± 5.8 (130-147) 139 ± 7.6 126 113 , -1.6) (1.2-1.5) 1.4 ± 0.1 (1.1-1.5) 1.3 ± 0.2 1.2 0.9 1-29.7) (13.9-20.6) 17.2 ± 2.5
m and in the form: mean ± s.d. (range). mean and in the form: m
μ
esocriconema xenoplax
M
are in All measurements
of Morphometrics
18.5-21.3 11.4-18.9 14.5-23.0 – 19.3-18.9 13.3-15.0 14.9-16.7 – – – 15.4 – – 18.8 6 ± 1.3 (4-7) (4-7) 6 ± 1.3 (1-4) 2 ± 1.3 (7-8) 8 ± 0.4 (8-9) 8 ± 0.6 (1-3) 2 ± 1.0 (5-7) 7 ± 1.0 (8-10) 8 ± 0.9 (3-4) 4 ± 0.4 (4-6) 5 ± 0.8 (8-9) 8 ± 0.5 (4-5) 4 ± 0.4 (3-5) 4 ± 0.8 (7-10) 9 ± 1.1 (3-5) 4 ± 0.8 (4-6) 5 ± 0.8 6 2 4 6 3 3 25± 0.8 (24-26) 25± 0.8 (24-26) (23-29) 27 ± 2.3 (14-19) 16 ± 1.9 (15-19) 17 ± 1.5 (14-16) 15 ± 0.8 18 14 (15-17) 16 ± 0.8 (27-34) 31 ± 3.0 (16-19) 17 ± 1.5 (31-33) 32 ± 1.2 (11-13) 12 ± 1.0 (10-20) 16 ± 4.3 (8-11) 10 ± 1.1 (20-22) 21 ± 1.0 (8-13) 10 ± 2.2 (17-20) 20 ± 2.2 11 21 9 – 98 ± 2.7 (95-102) (95-102) 98 ± 2.7 (97-103) 101 ± 2.5 (62-64) 63 ± 0.9 (60-63) 61 ± 1.6 (59-68) 62 ± 3.6 67 48 6.0 ± 0.3 (5.6-6.3) (5.6-6.3) 6.0 ± 0.3 (3.6-6.6) 4.7 ± 1.3 (8.6-10. 9.3 ± 0.6 1.0 ± 0.1 (0.7-1.0) (0.7-1.0) 1.0 ± 0.1 (1.0-1.1) 1.1 ± 0.1 (1.2 1.4 ± 0.2 140 ± 8.4 (135-155) (135-155) 140 ± 8.4 (101-154) 125 ± 20.4 (127- 133 ± 5.0 33.7 ± 5.8 (25.1-37.5) (25.1-37.5) 33.7 ± 5.8 (26.0-34.1) 30.5 ± 3.5 (25.1- 27.6 ± 2.0 15.3 ± 0.5 (14.5-15.8) (14.5-15.8) 15.3 ± 0.5 (8.8-13.5) 10.9 ± 2.1 (21.2- 22.3 ± 0.9 (24.8-27.6) 25.5 ± 1.2 (15.5-24.4) 20.1 ± 4.5 (28.5- 30.7 ± 1.9 21.0 ± 1.1 (19.5-22.6) (19.5-22.6) 21.0 ± 1.1 (12.4-19.8) 15.4 ± 3.2 (17.9- 20.6 ± 1.8 (27.0-31.1) 28.4 ± 1.6 22.7 ± 4.7(17.6-26.9) (33.2- 35.4 ± 1.8 13.3 ± 0.7 (13.1-14.3) (13.1-14.3) 13.3 ± 0.7 (7.6-15.3) 11.7 ± 3.1 (14.9- 17.6 ± 1.7 (13.1-35.5) 10.3 ± 28.0 (22.1-26.8) 23.6 ± 2.2 (18. 25.1 ± 3.7
Table 3 (continued). lip annulus body annulus lip annulus body annulus st nd st nd Pharynx length 1 Tail length Tail length Width at anus Annulus width diameter 2 diameter 2 diameter diameter diameter Spermatheca Spermatheca width R 1 Spermatheca length RSt Rpharynx Rex RV Rvan Ran VL/VB St%L
11 C.M. Etongwe et al.
Fig. 4. The 50% majority rule consensus tree obtained using Bayesian analysis of the COI mtDNA gene sequence alignment with the GTR+I+G model. Posterior probability of over 70% is given as a percentage for each appropriate clade. New sequences are indicated in bold. * – identified as Rotylenchus uniformis by Rybarczyk-Mydlowska et al. (2019); ** – identified as Helicotylenchus digonicus by Rybarczyk-Mydlowska et al. in the GenBank.
12 Molecular characterisation of plant-parasitic nematodes from Belgium Remark. Morphology and morphometrics of the Measurements are given in Table 3. specimen observed were very similar with that of Description. Female. Body cylindrical with, the original description (Loof & De Grisse, 1989) thick, retrorse annulus with rounded edges. Labial and other populations from Russia and Greece region oval shape, slightly elevated, submedian (Peneva et al., 2000; Karanastasi et al., 2008). lobes present and projecting anteriorly. Stylet robust, knobs anchor-shaped projecting anteriorly. Criconema annuliferum Hemizonid not seen. Anastomoses seen. (de Man, 1921) Micoletzky, 1925 (Fig. 6) Spermatheca round and empty, vulva slit-like with This species is known as a common species non-projected bulging lips. Tail plump with rounded found in many localities in Europe (Geraert, 2010). tip, terminal annulus bi-lobed. Females from three different populations were Male and juvenile. Not found. collected during this study from Zwijnaarde, Molecular characterisation. Intraspecific Kortrijk and Merendree. The morphological and sequence variation of C. informis was 0.5-3.1% (3- molecular data are presented in this study. 18 bp) and 4.7% (17 bp) for D2-D3 of 28S rRNA Measurements are given in Table 3. and COI genes, respectively. Phylogenetic positions Description. Female. Body annuli retrorse, of this species within some tylenchids are given in thick, rounded and with smooth edges. Anastomoses Figures 2, 3 and 4. In the D2-D3 28S rRNA gene seen in some specimens. First annulus collar-like, tree, this species clustered with Discocriconemella anteriorly directed, or straight, with second annuli sinensis (MK253537). narrower forming a collar-like appearance. Lips Remarks. The morphometrics and morphology of distinct and domed-shaped projecting anteriorly specimens collected during this study agree very well from the first annuli. Stylet strong with rounded with those of Geraert (2010) and Cordero et al. (2012). knobs. Vulva with overlapping lips, vagina sigmoid. Hemicycliophora thienemanni Anus located 3-6 annuli from the tail terminus. Tail (W. Schneider, 1925) Loos, 1948 conical ending with a rounded or truncate lobe (Voucher slides UGnem 242-243). This species was initially found inhabiting moist Male. Not found. soil in East Holstein, Germany by Schneider in 1925 Juveniles with collar like first annuli, body with and is known to inhabit a wide range of terrestrial as spines irregularly arranged in eight rows. well as aquatic habitats. It is extensively distributed Molecular characterisation. Intraspecific in Europe including Belgium (Subbotin et al., sequence variation for Criconema annuliferum was 2014). Males of this species are extremely rare and 0-1.1% (0-8 bp) and 0-1.9% (0-7 bp) for D2-D3 of Chitambar & Subbotin (2014) stated males have 28S rRNA and COI genes, respectively. been reported only from Ens, The Netherlands since Phylogenetic positions of this species within some its original description. A single female specimen of tylenchids are given in Figures 3 and 4. In the D2- this species was found in a sample from D3 28S rRNA gene tree, C. annuliferum formed a Blaarmeersen (Ghent). clade with Ogma decalineatus and Criconema Description. Female. Slender body, 921 μm demani and in the COI tree, this species clustered long, straight after fixation, cuticular sheath closely with C. longulum and O. murrayi. attached to inner one all along the body and loose Remark. The morphological and around the vulva. Cephalic region continuous, morphometrical data of the three populations lateral field with three longitudinal lines and few collected during this study are within the ranges of irregular breaks seen but no anastomoses observed. the original description as well as that of Peneva et Labial region conoid with distinct non-protruding al. (2000) from an oak forest in Russia. labial disc slightly elevated. Sclerotisation not observed on lip region. Stylet slender and straight, Criconemoides informis 88.5 μm long with rounded indistinct knobs that are (Micoletzky, 1922) Taylor, 1936 (Fig. 7) sloping posteriorly. Hemizonid not seen. Distance De Grisse (1963) first described this species as from excretory pore to anterior end 192.3 μm. C. flandriensis from sandy soil collected around Vulval lips unmodified with non-elongated vulval roots of unknown grass plants around a pear tree at sleeve. V = 86%. Spermatheca large, 22.1 μm long Eine (Marollen, Oudenaarde) in Belgium. Loof and empty. Tail 88 μm long, elongated and conoid (1965) later synonymised it with C. informis. The tapering abruptly in posterior one-third to a morphometric and molecular data of the populations distinctly offset narrower and elongated attenuated from Blaarmeersen (Ghent) and Merendree are distal portion with a narrowly rounded terminus. presented here. Tail annulation distinct.
13 C.M. Etongwe et al.
1150-1335
an ± s.d. (range).
Zwijnaarde m and in the form: me and in the form: m
μ
(17.9-25.5) (17.9-25.5) (20.1-22.4) 21.1 ± 1.1 28.1-31.2
4 (26.1-36.3) (29.4-34.8) 32.5 ± 2.8 23.3 .9 (50.0-109.5) .9 (50.0-109.5) (70.8-81.2) 73 ± 9.1 38.0 1.2 (3.4-6.6) (3.4-6.6) 1.2 (3.3-4.1) 3.8 ± 0.3 3.2-4.1 0.4 (7.6-8.9) (7.6-8.9) 0.4 (8.2-8.6) 8.4 ± 0.2 7.4-8.1 0.1 (0.5-0.8) (0.5-0.8) 0.1 (0.6-0.7) 0.7 ± 0.1 1.3 5.8 (31.1-46.9) (31.1-46.9) 5.8 (43.7-71.6) 52.3 ± 13.1 (41.2-45.6) 1.5 (41.4-49.0) 44.9 ± 3.4 33.1-47.9 34.1-40.5 0.6 (12.1-13.7) (12.1-13.7) 0.6 (42.2-48.6) 2.1 (12.9-14.9) 14.2 ± 0.9 (17.2-19.1) 0.7 (44.6-50.6) 47.7 ± 2.6 (22.7-25.9) 1.0 (18.1-24.4) 19.9 ± 2.4 (18.7-24.6) 21.4 ± 2.3 11.6-12.5 40.1-43.6 16.1-17.9 20.7-23.2 ± 0.6 (3.5-5.4) (3.5-5.4) ± 0.6 (5.1-7.4) 6.1-1.1 3.6-6.1 ± 0.6 (6.5-8.3) (6.5-8.3) ± 0.6 (6.9-8.6) 7.7 ± 0.7 5.9
(29.5-36.5) ± 2.4 (32.5-35.9) 34.1 ± 1.9 34.7
(1265-1570) 1435 ± 134.7 (1344-1748) 1565 ± 156.8
are in All measurements Belgium. from
females 1 male 8 females 10 females 3 males
R. robustus
and
– – – 42.2 – 42.2 – 19.2 – 43.2-44.3 – – 16.7-19.9 – – – – – – – (109-136) 122 ± 11.7 – 115-136
7 females 7 females 1 female 2 R. montanus R. robustus Destelbergen Garden Botanical University Ghent 20 ± 3.2 (16-22) (16-22) 20 ± 3.2 12 16; 18 (10-14) 12 ± 1.4 (9-13) 11 ± 1.7 – 58 ± 3.1 (53-60) (53-60) 58 ± 3.1 53 56 – (52-60) 55 ± 2.4 (55-57) 56 ± 0.7 – Rotylenchus montanus 7.4 ± 0.5 (7.0-8.1) (7.0-8.1) 7.4 ± 0.5 (2.4-2.6) 2.5 ± 0.1 – 5.5 5.4; 5.9 8.7 – 4.8 – – – 3.9 ± 0.4 (3.4-4.4) (3.4-4.4) 3.9 ± 0.4 4.4 3.7; 4.5 4.1 4.2 ± 6.7 ± 0.5 (6.5-7.4) (6.5-7.4) 6.7 ± 0.5 8.3 7.7; 8.4 6.8 8.2 ± 0.9 ± 0.1 (0.9-1.0) (0.9-1.0) 0.9 ± 0.1 0.8 0.8 1.2 0.7 ± 7.2 ± 0.3 (6.8-7.4) (6.8-7.4) 7.2 ± 0.3 7.6 8.0; 8.2 7.6 7.4 152 ± 8.0 (144-163) (144-163) 152 ± 8.0 – (143-161) 153 ± 8.4 179; 187 217 153 183; 196 (128-175) 157 ± 15.7 172 (139-191) 170 ± 21.6 (172-219) 194 ± 14.1 (176-225) 204 ± 20.2 145-150 168-199 28.9 ± 3.6 (22.8-34.7) (22.8-34.7) 28.9 ± 3.6 – – – – 27.6 ± 4.5 (24.0-34.2) (24.0-34.2) 27.6 ± 4.5 23.1 26.4 31.8 21.3 ± 4.1 44.4 ± 3.0 (41.3-48.1) (41.3-48.1) 44.4 ± 3.0 (28.1-33.3) 29.9 ± 2.4 45.0 30.5 42.9; 43.5 41.3 33.7 42.9 ± 26.1 31.5 ± 3. 22.2 ± 10.9 (7.1-31.0) (7.1-31.0) 22.2 ± 10.9 62.9 32.9; 43.5 32.9 39.1 ± 36.6 ± 1.2 (35.5-37.9) (35.5-37.9) 36.6 ± 1.2 (15.5-17.9) 16.6 ± 1.1 (15.6-16.5) 16.1 ± 0.4 43.7 19.0 20.3 47.8; 49.8 25.4; 25.7 18.7; 19.6 45.1 23.5 17.4 45.3 ± 18.0 ± 24.3 ± 12.6 ± 0.3 (12.4-12.9) (12.4-12.9) 12.6 ± 0.3 17.0 13.9; 14.6 13.4 13.1 ± 40.3 ± 5.0 (33.9-45.7) (33.9-45.7) 40.3 ± 5.0 71.3 55.9 41.2 70.0 ± 18 (23.6-25.4) 24.6 ± 0.8 36.6 33.9; 37.4 31.7 33.4 (1012-1200) 1096 ± 97.3 1647 1475; 1607 1309
Species
of Morphometrics
Table 4. Numberannuli of tail terminus Phasmid to tail Spicule length Gubernaculum length Tail length Tail length Maximum body diameter (MBD) Anal body diameter (ABD) Anterior end to secretory secretory to end Anterior pore excretory nerve to end Anterior ring to end Anterior pharyngeal gland end Pharynx overlapping Knobs width DGO Stylet length Conus length Shaft length Knob height Lip width V Lip height c c’ a b L N Characters Characters
14 Molecular characterisation of plant-parasitiic nematodes from Belgium
Fig. 5. Statistical parsimony network showing the phylogenetic relationships between ITS rRNA gene haplotypes. A: Hemicriconemoides pseudobrachyurus; B: Mesocriconema xenoplax and other Mesocriconema species; C: Rotylenchus robustus. Pies (circles) represent sequences of each species with the same hhaplotype and their size is proportional to the number of these sequences in the samples. Numbers of nucleotide differences between the sequences are indicated on lines connecting the pies. Small black circles represent missing haplottypes. New sequences are indicated in bold.
15 C.M. Etongwe et al. Male. Not found. that these sequences might not be correctly Molecular characterisation. Intraspecific identified and belong to R. montanus. sequence variation for this species was 0-0.7% (0-5 Remarks. The Belgian population of this bp) and 0.27% (0-1 bp) for D2-D3 of 28S rRNA and nematode agrees very well with those of the original COI genes, respectively. Phylogenetic position of description, except for some traits. Variations were this species within some tylenchids is given in observed in the position of the excretory pore Figures 3 and 4. The sequence of H. thienemanni whereby for some specimens it was located at the from Belgium clusters with sequences of this base of the pharynx as in the original description, or species from other countries in the D2-D3 28S posteriorly to the base of the pharynx. In addition, the rRNA gene tree. Belgian population is characterised by slightly longer Remarks. Morphology and morphometrics of body length (1012-1200 vs 977-1135 µm), slightly the specimen observed were very similar to that of longer stylet (35.5-37.9 vs 32.5-36.5 µm), wider other populations from different countries stylet knobs (7.0-8.1 vs 6-7 µm), broader maximum (Chitambar & Subbotin, 2014). diameter (41.3-48.1 vs 35.5-42.5 µm) and shorter anal body diameter (28.1-33.3 vs 24.5-30.5 µm). Rotylenchus montanus Vovlas, Subbotin, Troccoli, Liébanas & Castillo, 2008 (Fig. 8) Rotylenchus robustus (de Man, 1876) Filipjev, 1936 This nematode was originally described from apple orchards in Val di Non, Trento province, Italy De Man (1876) described this species from the (Vovlas et al., 2008). It is distinct from other Netherlands as Tylenchus robustus. It is Rotylenchus spp. based on the morphology of the lip characterised by a high, distinctly offset, region, tail shape, stylet length and vulva position. hemispherical cephalic region with 6-7 annuli, In Belgium, a population was collected around regularly areolated lateral fields at the pharyngeal grasses at the border of a ditch at Heusden region and regularly areolated lateral fields along (Destelbergen). This is the first record of the rest of the body, body without longitudinal R. montanus in Belgium. The morphological and striations, hemispherical tail. Later, Thorne (1949) molecular data of this population collected are described R. uniformis, a morphologically similar presented in this study. species. Loof & Oostenbrink (1958) synonymised R. Measurements are given in Table 4. uniformis with R. robustus and Krall (1978) and Description. Body habitus spiral after fixation. Siddiqi (2000) accepted this synonymisation. Labial framework well developed and hemispherical However, Seinhorst (1991) stated that these species with six to seven annuli. Stylet robust with shaft are morphologically different and listed some equal length as the conus, basal knobs rounded. In differential characters. Several other authors also some specimens, the excretory pore is located at the differentiated these species from each other. Brzeski level, or posterior to the end of the pharynx. (1998) distinguished R. robustus from R. uniformis Pharyngeal glands, overlapping intestine dorsally, based on stylet length (less than 40 µm vs 40 µm or relatively long with one large nucleus seen. more) and lateral field areolation near vulva (absent Hemizonid not seen. Lateral field with four lateral vs present). Castillo & Vovlas (2005) recognised lines, regularly areolated at the pharyngeal region both species and considered that R. robustus could and smooth along the rest of the body with four be differentiated from R. uniformis by a higher lateral lines present. Reproductive system didelphic number of lip annuli (6-8 vs 5); lateral fields with well-developed genital branches. Spermatheca areolated in pharyngeal region and irregularly spherical and empty. Vulva of some specimens with areolated at mid-body vs areolated only in protruding epiptygma. Phasmid pore-like, and pharyngeal region; and female tail hemispherical vs anterior to anus. Tail rounded, regularly annulated rounded. In the present paper, we consider these with depression seen in some specimens. species as co-specific as has been proposed by Loof Male. Not found. & Oostenbrink (1958). Molecular characterisation. Intraspecific Measurements are given in Table 4. sequence variation for this species was 0-0.1% (0-1 Description. Female. Habitus spiral or C- bp) for D2-D3 of 28S rRNA gene. Phylogenetic shaped, lateral fields areolated at the pharyngeal positions of this species within some tylenchids are region and irregularly along the whole body, labial given in Figures 2, 3 and 4. In the 18S rRNA gene region hemispherical, distinctly offset with 6-7 tree, the sequence of R. montanus clustered with two distinct annuli and a terminal disc. Stylet well- sequences identified as R. robustus. We assumed developed with rounded knobs, long pharyngeal glands
16 Molecular characterisation of plant-parasitiic nematodes from Belgium
Table 5. Morphometrics of Helicotylenchus varicaudatus from Belgium. All measurements are in μm and in the form: mean ± s.d. (range).
Citadel park, Character Blaarmeersen Ghent N 4 females 1 female 1 female 1 female L 785 ± 26 (758-810) 683 709 662 a 25.4 ± 0.7 (24.6-28.8) 27.4 23.4 22.9 b’ 4.9 ± 0.3 (4.5-5.2) 4.6 5.0 4.9 c 52.2 ± 2.6 (50.4-54.0) 53.0 66.3 49 c’ 0.8 ± 0.1 (0.7-0.9) 0.8 0.6 0.8 o 25.9 ± 3.8 (25.1-27.6) 25.6 26.0 27.8 V 61 ± 1.0 (61-62) 62 62 58 Lip height 4.2 ± 0.1 (4.1-4.2) 4.3 5.4 3.2 Lip diameter 7.6 ± 0.6 (6.9-8.0) 8.8 8.0 7.9 Stylet length 31.7 ± 0.9 (30.7-32.3) 31.7 30.5 24.0 Conus length 14.5 ± 1.4 (12.8-15.8) 15.4 15.0 9.8 DGO 8.2 ± 0.5 (7.7-8.9) 8.1 7.9 6.7 m 45.7 ± 3.1 (41.7-48.9) 48.6 49.0 40.7 Pharynx length 157 ± 7.7 (148-167) 148 143 141 Anterior end to pharyngeal 27.8 ± 0.7 (27.0-28.5) – 22.1 34.3 gland end Anterior end to excretory 121 ± 10.2 (110-1344) 110 121 104 pore Maximum body diameter 29.4 ± 3.4 (24.9-32.9) 24.9 30.4 28.9 Lateral field width 2.1 ± 0.3 (1.8-2.3) 1.8 1.7 1.6 Tail length 14.2 ± 1.4 (12.9-15.6) 12.9 10.7 13.5 Anal body diameter 17.7 ± 2.2 (15.6-20.0) 15.6 16.5 16.5 Tail annuli number 9 ± 1.4 (8-10) – 10 12
Fig. 6. LM photos of Criconema annuliferum. A-D: Anterior region variations of femaales; E: Anastomoses; F-G: Posterior region of females; H: Anterior region of juvenile.
17 C.M. Etongwe et al.
Fig. 7. LM photos of Criconemoides informis. A: Anterior region; B: Posterior region. overlapping intestine dorsally. Spermatheca rounded Helicotylenchus varicaudatus and packed with sperm cells. Tail hemispherical, Yuen, 19644 (Fig. 9) distinctly and regularly annulated, phasmid distinct above the anus and pore-like. This species was described from Broadbalk Male. Habitus open C shape with a labial region Wilderness, Harpenden, England (Yuen, 1964). It more distinctly offset and elevated. has a very variable tail moorrphology and is distinct Molecular characterisation. Intraspecific from the others based on its stylet length, number of sequence variation for R. robustus was 0-0.5% (0-4 head annuli and the posittion of phasmids. The bp), 0.2-0.8% (1-4 bp), 0.7-17.8% (1-70 bp) for 18S morphological and molecular data are given for rRNA, D2-D3 of 28S rRNA and COI genes, samples collected from Blaarmeersen and the respectively. Phylogenetic positions of this species Citadel Park (Ghent). within some tylenchids is given in Figures 2, 3 and Measurements are given in Table 5. 4. The ITS gene alignment of R. robustus included Description. Female. Body habitus spiral or C- seven sequences with three new sequences obtained shaped. Lip region hemisppherical and continuous, from this study and it was 1019 bp long. Belgian with indistinct lip annuli. Stylet slender with flat sequences of this species clustered with other knobs. Hemizonid not seen. Excretory pore R. robustus/uniformis populations from GenBank located anterior to level of pharyngo-intestinal with a maximum intraspecific variation of 0.2-3.1% valve. Pharyngeal gland overlapping intestine (1-17 bp) (Fig. 5C). ventrally. Lateral field with four lines, areolated
18 Molecular characterisation of plant-parasitiic nematodes from Belgium
Fig. 8. LM photos of Rotylenchus montanus. A, E: Anterior region; B: Areolation at pharrynx region; C-D: Posterior region; F: Vulva region; G: Total body. at the pharyngeal region. Spermatheca offset, P. bukowinensis. Raski (1975) re-described the rounded, and empty. Epiptygma folded into vagina. species and validated it as a valid species. In this Phasmids pore-like, located posterior to anus. Tail study, populations from ZZwijnaarde and Ghent shape is very variable. University Botanical garden have been molecularly Molecular characterisation. Intraspecific and morphologically analysed. sequence variation was 0.7-1.9% (4-22 bp) and 0- Measurements are given in Table 6. 7.4% (0-29 bp) for D2-D3 of 28S rRNA and COI Description. Body slighttly curved ventrally. Lip genes, respectively. Phylogenetic positions of this region conically rounded and continuous with a species within some tylenchids are given in Figures weak framework. Stylet long and slender. Excretory 3 and 4. pore located between the position of the basal Remarks. The morphology and morphometrics pharyngeal bulb or isthmus. Hemizonid seen, lateral of the Belgian populations of this nematode agree field with four lines, lateral vulva flaps distinct. very well with those of the original description and Spermatheca large, and round, and filled with sperm other literature (Yuen, 1964; Loof, 1984; Yeates & cells. Tail with finely rounded tip. Wouts, 1992; Brzeski, 1998; Bert & Geraert, 2000; Molecular characterisaattion. The D2-D3 of 28S Reis et al., 2010). rRNA gene sequences of Belgian populations were identical with those of P. nanus type A from Paratylenchus nanus California, USA (Van den Berg et al., 2014a). Cobb, 1923 (Figs 10A & B) Phylogenetic positions of this species within some This species was described by Cobb (1923) from tylenchids are given in Figures 2, 3 and 4. a single female collected near Devil’s Lake in North Remark. The morphology and morphometrics of Dakota, USA. Geraert (1965) proposed the specimens from this study agree with that of P. nanus synonymisation of this species with type A reported by Van den Berg et al. (2014a).
19 C.M. Etongwe et al.
Table 6. Morphometrics of Paratylenchus nanus and Paratylenchus sp. F from Belgium. All measurements are in μm and in the form: mean ± s.d. (range). Species Paratylenchus nanus Paratylenchus sp. F
Characters Ghent University Merendree Zwijnaarde Botanical garden N 3 females 5 females 3 females L 340-364 356 ± 28.4 (313-389) 330-350 a 21.2-22.0 19.4 ± 2.2 (17.1-22.0) 19.4-20.8 b 3.5-3.8 4.1 ± 0.3 (3.7-4.4) 3.7-4.0 c 14.9-17.4 12.7 ± 0.9 (11.4-13.4) 13.8-15.4 c’ 1.9-2.3 2.8 ± 0.3 (2.4-3.0) 2.1-2.5 o 12.2-14.0 25.0 ± 1.5 (23.6-26.7) 12.2-15.7 DGO 3.6-4.1 7.1 ± 0.4 (6.5-7.7) 3.6-4.6 V 83-84 82 ± 1.1 (80-83) 81-83 Stylet length 29.5-29.9 28.3 ± 1.1 (26.7-29.6) 27.9-29.7 Metenchium length 19.2-20.6 18.1 ± 1.0 (16.7-19.2) 19.9-21.8 Telenchium length 7.5-8.4 7.7 ± 1.8 (5.8-10.0) 5.6-6.5 m 65.0-69.0 64.3 ± 4.6 (57.7-69.3) 71.6-74.7 Stylet knob height 1.8-2.1 2.5 ± 0.2 (2.2-2.8) 1.5-1.8 Stylet knob width 4.1-5.0 4.3 ± 0.2 (4.0-4.6) 3.9-4.9 Pharynx length 89.1-98.7 85.8 ± 3.2 (81.1-88.7) 86.1-91.2 Anterior end to excretory pore 75.3-76.9 75.2 ± 4.1 (70.3-79.1) 71.2-78.0 Diam at mid body 15.9-17.1 18.4 ± 1.3 (16.6-20.2) 16.7-17.0 Lip region diam 6.2-6.9 6.7 ± 0.2 (6.4-6.9) 6.1-6.8 Lip region height 3.1-3.8 3.1 ± 0.3 (2.8-3.5) 3.4-3.9 Annulus width 1.4-1.7 1.3 ± 0.2 (1.1-1.5) 1.2-1.9 Tail length 20.2-22.8 28.8 ± 1.7 (27.3-30.4) 22.3-25.3 EP%L 20.8-22.2 21.2 ± 0.8 (20.3-22.5) 21.0-22.3 V-anus distance 30.0-33.3 37.9 ± 2.5 (35.5-40.3) 30.0-38.5 Spermatheca length 12.4-18.8 12.1 ± 2.0 (10.8-15.0) 16.9-14.6 Spermatheca diameter 9.9-11.0 9.5 ± 1.6 (7.8-11.5) 10.0-11.5 PUB length – 17.4 – St%L 8.1-8.8 8 ± 0.3 (7.3-8.5) 8.0-9.0
Molecular characterisation. Intraspecific Paratylenchus sp. F sequence variation was 0-0.2% (0-2 bp), 0.6% (Figs 10C & D) (4 bp) and 0-0.5% (0-2 bp) for 18S rRNA, for D2- This nematode was found in samples collected in D3 of 28S rRNA and COI genes, respectively. Blaarmeersen and Merendree and was not identified Phylogenetic positions of this species within some at the species level and provisionally named as tylenchids are given in Figures 3 and 4. species F here. Amplimerlinius icarus Measurements are given in Table 6. Wallace & Greet, 1964 (Fig. 11) Description. Female. Body slender and relatively small. Cephalic region rounded to faintly This nematode was described from grassland in truncate. Submedian lobes absent or indistinct, Winches Farm, St. Albans, England (Wallace & excretory pore located at the level of the basal Greet, 1964). In this study, a population of this pharyngeal bulb. Stylet short, spermatheca oval and species from Eine (Oudenaarde) has been empty. Tail finely rounded, one specimen seen with morphologically and molecularly characterised. digitate tail. Vulval flap rounded. Measurements are given in Table 7. Male. Not found. Description. Female. Body spiral or C-shaped Juvenile very similar to female except for the after fixation. Lip region conoid and continuous. absence of a stylet.
20 Molecular characterisation of plant-parasitiic nematodes from Belgium
Fig. 9. LM photos of Helicotylenchus varicaudattuus. A: Anterior region; B-G: Posterior region showing variations in tail shape. Scales = 25 µm.
Table 7. Morphometrics of Amplimerlinius carus from Belgium. All measurements are iin μm and in the form: mean ± s.d. (range).
Species Amplimerlinius icarus
Characters Eine Geraert (2011) Wallace & Greet (1964) N 4 femalees ? 20 females L 1387 ± 23.8 (1358-1416) 1450-2010 1450-1960 a 29.8 ± 1.4 (29.00-31.2) 29-39 29-34 b 7.3 ± 1.9 (6.2--10.1) – 5.9-6.9 c 21.3 ± 1.3 (19.44-22.3) 18-25 19-25 c’ 2.0 ± 0.1 (1.9-2.2) 1.8-2.7 – V 54 ± 2.2 (51-57) 50-58 50-57 Number of head annuli 5 ± 3.3 (5-8) 4-6 8 Lip height 6.2 ± 0.5 (5.5-6.5) – – Lip width 12.2 ± 1.0 (10.99-13.1) – – Stylet length 37.5 ± 2.2 (34.44-39.4) 34-42 34-42 Conus length 20.4 ± 1.8 (17.77-21.8) – – Shaft length 17.1 ± 0.7 (16.66-18.2) – – Knob height 4.3 ± 0.3 (3.9-4.7) – – DGO 4.6 ± 1.9 (3.7-5.4) 4-5 – Anterior end to secretory 160 ± 8.1 (151-170) – – excretory pore Anterior end to nerve ring 143-156 – – Anterior end to pharyngeal gland 199 ± 43.1 (1335-225) 223-299 – end Maximum body diameter (MBD) 46.5 ± 1.9 (44.44-48.9) 45-58 – Anal body diameter (ABD) 32.3 ± 0.7 (31.55-33.1) – – Tail length 65.4 ± 4.4 (61.00-71.6) 65-93 – Number of tail annuli 49 ± 4.1 (45-54) 34-57 50-59 Phasmid position (posterior to the 37.8-40.0 24-44 – anus)
21 C.M. Etongwe et al.
Fig. 10. LM photos of Paratylenchus nanus. A: Anterior region; B: Posterior region; LM photos of Paratylenchus sp. F. C: Anterior region; D: Posterior region.
Fig. 11. LM photos of Amplimerlinius icarus. A: Female and male; B: Anterior region of female; C: Posterior region of female; D: Posterior region of male. B, C, D have same scale.
Labial framework heavily sclerotised with 5-8 annuli. 0.2% (1 bp) for D2-D3 of 28S rRNA and up to Stylet robust with round knobs projecting posteriiorly. 12.7% (50 bp) for COOII genes. Phylogenetic Excretory pore seen opening at the level of the positions of this species witthin some tylenchids are isthmus of the pharynx. Hemizonid located slightly given in Figures 2, 3 and 4. above or same position as the opening of the Remark. Morphology and morphometrics of the excretory pore. Lateral fields present with six lines, specimen observed were very similar with that of with irregular areolations at the pharyngeal and tail the original description of Wallace & Greet (1964) regions. Deirids not seen. Basal pharyngeal bulb and that of Geraert (20011). However, some ovoid, and does not overlap with the intestine. Vulva variations were observed ass specimens in this study located approximately at the middle of the body. had shorter total body length (1387 ± 23.8 (1358- Spermatheca round with spermatozoids present. Tail 1416) vs 1450-2010) and no vulva lips were seen as cylindrical, annulated, with broadly rounded described by Geraert (2011). terminus, in some specimens. Phasmid prominent. Sequence and phylogenetic analysis. The 18S Male. Not found. rRNA gene alignment inclluuded 85 sequences and Molecular characterisation. Intraspecific two sequences selected as outgroups from the sequence variation was 0.3% (3 bp) for 18S rRNA, genera and was 896 bp iin length. Phylogenetic
22 Molecular characterisation of plant-parasitic nematodes from Belgium analysis resulted in a majority consensus BI tree BERT, W. & GERAERT, E. 2000. Nematode species of the with four major moderately supported clades order Tylenchida, new to the Belgian nematofauna (Fig. 2). with additional morphological data. Belgian Journal The D2-D3 of 28S rRNA gene alignment of Zoology 130: 47-57. included 112 sequences, including two sequences BERT, W., COOMANS, A., CLAERBOUT, F., GERAERT, E. & selected as outgroup taxa and was 781 bp in length. BORGONIE, G. 2003. Tylenchomorpha (Nematoda: Phylogenetic analysis resulted in majority consensus Tylenchina) in Belgium, an updated list. Nematology BI tree with three major supported clades (Fig. 3). 5: 435-440. The COI gene alignment included 88 sequences BERT, W., LELIAERT, F., VIERSTRAETE, A.R., including two sequences selected as outgroups and VANFLETEREN, J.R. & BORGONIE, G. 2008. Molecular was 393 bp in length. Phylogenetic analysis resulted phylogeny of the Tylenchina and evolution of the in a majority consensus BI tree with three major female gonoduct (Nematoda: Rhabditida). Molecular clades (Fig. 4). Phylogenetics and Evolution 48: 728-744. DOI: 10.1016/j.ympev.2008.04.011 ACKNOWLEDGEMENTS BHADURY, P., AUSTEN, M.C., BILTON, D.T., LAMBSHEAD, P.J.D., ROGERS, A.D. & SMERDON, G.R. 2006. The authors thank Marjolein Couvreur Development and evaluation of a DNA-barcoding (Nematology Research Unit, Department of approach for the rapid identification of nematodes. Biology, Ghent University) for her assistance during Marine Ecology Progress Series 320: 1-9. several aspects of this project, especially scanning DOI: 10.3354/meps320001 electron microscopy; and Prof. André De Grisse for BRZESKI, M.W. 1998. Nematodes of Tylenchina in his help to retrieve nematode type localities. Poland and Temperate Europe. Poland, Muzeum Catherine Malike Etongwe was supported by a Instytut Zoologii Polska Akademia Nauk. 397 pp. VLIR-UOS ICP scholarship. CANTALAPIEDRA-NAVARRETE, C., NAVAS-CORTES, J.A., LIEBANAS, G., VOVLAS, N., SUBBOTIN, S.A., REFERENCES PALOMARES-RIUS, J.E. & CASTILLO, P. 2013. Comparative molecular and morphological ALTUN AFSHAR, F.J., POURJAM, E. & PEDRAM, M. 2019. characterisations in the nematode genus Rotylenchus: New morphological observations on Neolobocriconema Rotylenchus paravitis n. sp., an example of cryptic serratum (Khan & Siddiqi, 1963) Mehta & Raski, 1971 speciation. Zoologischer Anzeiger – A Journal of (Rhabditida: Criconematidae). Nematology 21: 419-434. Comparative Zoology 252: 246-268. DOI: 10.1163/15685411-00003223 DOI: 10.1016/j.jcz.2012.08.002 AHMED, M., SAPP, M., PRIOR, T., KARSSEN, G. & BACK, CASTILLO, P. & VOVLAS, N. 2005. Bionomics and M.A. 2016. Technological advancements and their Identification of the Genus Rotylenchus (Nematoda: importance for nematode identification. Soil 2: 257- Hoplolaimidae). Nematology Monographs and 270. DOI: 10.5194/soil-2-257-2016 Perspectives, Volume 3. The Netherlands, Brill AMIRI, S., SUBBOTIN, S.A. & MOENS, M. 2003. Academic Publishers. 378 pp. Comparative morphometrics and RAPD studies of CHITAMBAR, J.J. & SUBBOTIN, S.A. 2014. Systematics of Heterodera schachtii and H. betae populations. the Sheath Nematodes of the Superfamily Russian Journal of Nematology 11: 87-95. Hemicycliophoroidea. Nematology Monographs and AZIZI, K., ESKANDARI, A., KAREGAR, A., GHADERI, R., Perspectives, Volume 10 (Series Eds: D.J. Hunt & VAN DEN ELSEN, S., HOLTERMAN, M. & HELDER, J. R.N. Perry). The Netherlands, Brill Academic 2016. Morphological and molecular data support the Publishers. 732 pp. monophyletic nature of the genus Pratylenchoides COBB, N.A. 1923. Notes on Paratylenchus, a genus of Winslow, 1958 (Nematoda: Merliniidae) and reveal nemas. Journal of the Washington Academy of its intrageneric structuring. Nematology 18: 1165- Sciences 13: 254-257. 1183. DOI: 10.1163/15685411-00003023 CONSOLI, E., AKANWARI, J. & SUBBOTIN, S.A. 2017. BAE, C.H., SZALANSKI, A.L. & ROBBINS, R.T. 2009. Morphological and molecular characterisation of Phylogenetic analysis of the Hoplolaiminae inferred Paratrophurus bursifer (Loof, 1960) Siddiqi, 1971 from combined D2 and D3 expansion segments of (Nematoda: Tylenchida) from Belgium. Russian 28S rRNA. Journal of Nematology 41: 28-34. Journal of Nematology 25: 17-22. BANDELT, H., FORSTER, P. & RÖHL, A. 1999. Median- COOMANS, A. 1989. Overzicht van de vrijlevende joining networks for inferring intraspecific nematofauna van België. Verhandeligen van het phylogenies. Molecular Biology and Evolution 16: Symposium “Invertebraten van België”, November 37-48. DOI: 10.1093/oxfordjournals.molbev.a026036 25-26, 1988, Brussels, Belgium. 43-56.
23 C.M. Etongwe et al.
CORDERO, M.A., ROBBINS, R.T. & SZALANSKI, A.L. GERAERT, E. 2011. The Dolichodoridae of the World. 2012. Taxonomic and molecular identification of Identification of the Family Dolichodoridae Bakernema, Criconema, Hemicriconemoides, Ogma (Nematoda). Belgium, Academia Press. 520 pp. and Xenocriconemella species (Nematoda: GERMANI, G. & ANDERSON, R.V. 1991. Taxonomic notes Criconematidae). Journal of Nematology 44: 427-446. on some Hemicriconemoides species and description CURRAN, J., DRIVER, F., BALLARD, J.W.O. & MILNER, of a new species. Journal of Nematology 23: 502-510. R.J. 1994. Phylogeny of Metarhizium: analysis of GHADERI, R., KAREGAR, A., NIKNAM, G. & SUBBOTIN, ribosomal DNA sequence data. Mycological Research S.A. 2014. Phylogenetic relationships of 98: 547-555. DOI: 10.1016/S0953-7562(09)80478-4 Telotylenchidae Siddiqi, 1960 and Merliniidae DAMME, N., WAEYENBERGE, L. & VIAENE, N. 2013. First Siddiqi, 1971 (Nematoda: Tylenchida) from Iran, as report of the root-knot nematode Meloidogyne inferred from the analysis of the D2-D3 expansion artiellia in Belgium. Plant Disease 97: 152. fragments of 28S rRNA gene sequences. Nematology DOI: 10.1094/PDIS-91-7-0908B 16: 863-877. DOI: 10.1163/15685411-00002815 DE GRISSE, A.T. 1963. Criconemoides flandriensis n. sp. GOLHASAN, B., HEYDARI, R., ÁLVAREZ-ORTEGA, S., (Nematoda: Criconematidae). Nematologica 9: 547-552. MECKES, O., PEDRAM, M. & ATIGHI, M.R. 2016. DE GRISSE, A.T. 1964. Hemicriconemoides Rotylenchus sardashtensis n. sp., a monosexual pseudobrachyurum n. sp. (Nematoda: species from Iran, with molecular identification and Criconematidae). Nematologica 10: 369-372. detailed morphological observations on an Iranian DE GRISSE, A.T. 1969. Redescription ou modifications de population of Rotylenchus cypriensis Antoniou, 1980 quelques techniques utilisées dans l’étude des nématodes (Nematoda: Rhabditida: Hoplolaimidae). Systematic phytoparasitaires. Mededelingen van de Rijksfakulteit Parasitology 93: 395-411. DOI: 10.1007/s11230-015- Landbouwwetenschappen Gent 34: 351-369. 9619-3 DE MAN, J.G. 1876. Onderzoekingen over vrij in de aarde GONÇALVES DE OLIVEIRA, C.M., ROCHA MONTEIRO, A. levende Nematoden. Tijdschrift der Nederlandsche & BLOK, V.C. 2011. Morphological and molecular Dierkundige Vereeniging 2: 78-196. diagnostics for plant-parasitic nematodes: working DERYCKE, S., VANAVERBEKE, J., RIGAUX, A., together to get the identification done. Tropical Plant BACKELJAU, T. & MOENS, T. 2010. Exploring the use Pathology 36: 65-73. DOI: 10.1590/S1982- of cytochrome oxidase c subunit 1 (COI) for DNA 56762011000200001 barcoding of free-living marine nematodes. PLoS One HEBERT, P.D.N., RATNASINGHAM, S. & DEWAARD, J.R. 5: e13716. DOI: 10.1371/journal.pone.0013716 2003. Barcoding animal life: cytochrome c oxidase ESMAEILI, M., HEYDARI, R., CASTILLO, P., BIDHENDI, subunit 1 divergences among closely related species. M.Z. & PALOMARES-RIUS, J.E. 2016. Molecular Proceedings of the Royal Society B, Biological characterization of two known species of Sciences 270 (supplementary 1): S96-S99. Paratylenchus Micoletzky, 1922 from Iran with notes DOI: 10.1098/rsbl.2003.0025 on the validity of Paratylenchus audriellus Brown, HOLTERMAN, M., VAN DER WURFF, A., VAN DEN ELSEN, 1959. Nematology 18: 591-604. S., VAN MEGEN, H., BONGERS, T., HOLOVACHOV, O., DOI: 10.1163/15685411-00002979 BAKKER, J. & HELDER, J. 2006. Phylum-wide analysis EYUALEM, A. & BLAXTER, M. 2003. Comparison of of SSU rDNA reveals deep phylogenetic relationships biological, molecular, and morphological methods of among nematodes and accelerated evolution toward species identification in a set of cultured crown clades. Molecular Biology and Evolution 23: Panagrolaimus isolates. Journal of Nematology 35: 1792-1800. DOI: 10.1093/molbev/msl044 119-128. HOLTERMAN, M., KARSSEN, G., VAN DEN ELSEN, S., VAN FERRI, E., BARBUTO, M., BAIN, O., GALIMBERTI, A., UNI, MEGEN, H., BAKKER, J. & HELDER, J. 2009. Small S., GUERRERO, R., FERTE, H., BANDI, C., MARTIN, C. subunit rDNA-based phylogeny of the Tylenchida & CASIRAGHI, M. 2009. Integrated taxonomy: sheds light on relationships among some high-impact traditional approach and DNA barcoding for the plant-parasitic nematodes and the evolution of plant identification of filarioid worms and related parasites feeding. Phytopathology 99: 227-235. (Nematoda). Frontiers in Zoology 6: 1-12. DOI: 10.1094/PHYTO-99-3-0227 DOI: 10.1186/1742-9994-6-1 JANSSEN, T., KARSSEN, G., COUVREUR, M., GERAERT, E. 1965. The genus Paratylenchus. Nematologica WAEYENBERGE, L. & BERT, W. 2017A. The pitfalls of 11: 301-334. DOI: 10.1163/187529265X00221 molecular species identification: a case study within GERAERT, E. 2010. The Criconematidae of the World. the genus Pratylenchus (Nematoda: Pratylenchidae). Identification of the Family Criconematidae Nematology 19: 1179-1199. DOI: 10.1163/15685411- (Nematoda). Belgium, Academia Press. 615 pp. 00003117
24 Molecular characterisation of plant-parasitic nematodes from Belgium
JANSSEN, T., KARSSEN, G., ORLANDO, V., SUBBOTIN, S.A. implications for biodiversity, biogeography and the & BERT, W. 2017B. Molecular characterization and evolution of hermaphroditism. BMC Evolutionary species delimiting of plant-parasitic nematodes of the Biology 7: 104. genus Pratylenchus from the penetrans group MELDAL, B.H.M., DEBENHAM, N.J., DE LEY, P., DE LEY, (Nematoda: Pratylenchidae). Molecular Phylogenetics I.T., VANFLETEREN, J.R., VIERSTRAETE, A.R., BERT, and Evolution 117: 30-48. W., BORGONIE, G., MOENS, T., TYLER, P.A., AUSTEN, DOI: 10.1016/j.ympev.2017.07.027 M.C., BLAXTER, M.L., ROGERS, A.D. & LAMBSHEAD, JENKINS, W.R. 1964. A rapid centrifugal-flotation method P.J.D. 2007. An improved molecular phylogeny of the for separating nematodes from soil. Plant Disease Nematoda with special emphasis on marine taxa. Reporter 48: 692. Molecular Phylogenetics and Evolution 42: 622-636. KANZAKI, N. & FUTAI, K. 2002. A PCR primer set for DOI: 10.1016/j.ympev.2006.08.025 determination of phylogenetic relationships of MILLER, S.E. 2007. DNA barcoding and the renaissance Bursaphelenchus species within the xylophilus group. of taxonomy. PNAS 104: 4775-4776. Nematology 4: 35-41. DOI: 10.1163/ DOI: 10.1073/pnas.0700466104 156854102760082186 NGUYEN, H.T., TRINH, Q.P., COUVREUR, M., SINGH, P.R., KARANASTASI, E., HANDOO, Z.A. & TZORTZAKAKIS, E.A. DECRAEMER, W. & BERT, W. 2019A. Molecular and 2008. First report of Mesocriconema xenoplax morphological characterisation of a new root-lesion (Nematoda: Criconematidae) in Greece and first nematode, Pratylenchus horti n. sp. record of Viburnum sp. as a possible host for this ring (Tylenchomorpha: Pratylenchidae), from Ghent nematode. Helminthologia 45: 103-105. University Botanical Garden. Nematology 21: 739- DOI: 10.2478/s11687-008-0019-y 752. DOI: 10.1163/15685411-00003249 KRALL, E.L. 1978. [Root Parasitic Nematodes. Family NGUYEN, H.T., TRINH, Q.P., COUVREUR, M., SINGH, P.R., Hoplolaimidae.] USSR, Nauka. 420 pp. (in Russian). DECRAEMER, W. & BERT, W. 2019B. First Report of LOOF, P.A.A. 1965. Zur Taxonomie von Criconemoides Scutellonema brachyurus (Steiner, 1938) Andrassy, rusticus (Micoletzky) und C. informis (Micoletzky). 1958 and occurrence of Meloidogyne incognita Mitteilungen aus dem Museum für Naturkunde in (Kofoid & White, 1919) Chitwood, 1949 in Belgium. Berlin. Zoologisches Museum und Institut für Journal of Nematology 51: e2019-62. Spezielle Zoologie (Berlin) 41: 183-192. DOI: 10.21307/jofnem-2019-062 DOI: 10.1002/mmnz.4830410206 NGUYEN, H.T., TRINH, Q.P., COUVREUR, M., SINGH, P.R., LOOF, P.A.A. 1984. Observations on Helicotylenchus DECRAEMER, W. & BERT, W. 2019C. Description of varicaudatus Yuen, 1964 (Nematoda, Rotylenchus rhomboides n. sp. and a Belgian Hoplolaimidae). Mededelingen – Faculteit population of Rotylenchus buxophilus Landbouwkundige en Toegepaste Biologische (Tylenchomorpha: Hoplolaimidae). Journal of Wetenschappen, Universiteit Gent 49: 1-7. Nematology 51: 1-20. DOI: 10.21307/jofnem-2019-023 LOOF, P.A.A. & DE GRISSE, A. 1989. Taxonomic and NORUZI, E., ASGHARI, R., ATIGHI, M.R., ESKANDARI, A., nomenclatorial observations on the genus CANTALAPIEDRA-NAVARRETE, C., ARCHIDONA- Criconemella De Grisse and Loof, 1965 sensu Luc YUSTE, A., LIEBANAS, G., CASTILLO, P. & and Raski, 1981. Mededelingen – Faculteit PALOMARES-RIUS, J.E. 2015. Description of Landbouwkundige en Toegepaste Biologische Rotylenchus urmiaensis n. sp. (Nematoda: Wetenschappen, Universiteit Gent 54: 53-74. Hoplolaimidae) from North-western Iran with a LOOF, P.A.A. & OOSTENBRINK, M. 1958. The identity of molecular phylogeny of the genus. Nematology 17: Tylenchus robustus De Man. Nematologica 3: 34-43. 607-619. DOI: 10.1163/15685411-00002895 MARIA, M., CAI, R., CASTILLO, P. & ZHENG, J. 2018. OLSON, M., HARRIS, T., HIGGINS, R., MULLIN, P., Morphological and molecular characterisation of POWERS, K., OLSON, S. & POWERS, T.O. 2017. Hemicriconemoides paracamelliae sp. n. (Nematoda: Species delimitation and description of Criconematidae) and two known species of Mesocriconema nebraskense n. sp. (Nematoda: Hemicriconemoides from China. Nematology 20: 403- Criconematidae), a morphologically cryptic, 422. DOI: 10.1163/15685411-00003147 parthenogenetic species from North American MARIA, M., CAI, R., SUBBOTIN, S.A. & ZHENG, J. 2019. Grasslands. Journal of Nematology 49: 42-66. Description of Discocriconemella sinensis n. sp. DOI: 10.21307/jofnem-2017-045 (Nematoda: Criconematidae) from the rhizosphere of ORTIZ, V., PHELAN, S. & MULLINS, E. 2016. A temporal Camellia sinensis in China. Nematology 21: 779-792. assessment of nematode community structure and DOI: 10.1163/15685411-00003252 diversity in the rhizosphere of cisgenic Phytophthora MAYER, W.E., HERRMANN, M. & SOMMER, R.J. 2007. infestans-resistant potatoes. BMC Ecology 16: 55. Phylogeny of the nematode genus Pristionchus and DOI: 10.1186/s12898-016-0109-5
25 C.M. Etongwe et al.
PALOMARES-RIUS, J.E., CANTALAPIEDRA-NAVARRETE, QING, X., PEREIRA, T. J., SLOS, D., COUVREUR, M. & C., ARCHIDONA-YUSTE, A., SUBBOTIN, S.A. & BERT, W. 2018. A new species of Malenchus CASTILLO, P. 2017. The utility of mtDNA and rDNA (Nematoda: Tylenchomorpha) with an updated for barcoding and phylogeny of plant-parasitic phylogeny of the Tylenchidae. Nematology 20: 815- nematodes from Longidoridae (Nematoda, Enoplea). 836. DOI: 10.1163/15685411-00003177 Scientific Reports 7: 1-42. DOI: 10.1038/s41598-017- QING, X., WANG, M., KARSSEN, G., BUCKI, P., BERT, W. 11085-4 & BRAUN-MIYARA, S. 2020. PPNID: a reference PANAHANDEH, Y., POURJAM, E., ATIGHI, M.R. & database and molecular identification pipeline for PEDRAM, M. 2014. Morphological and molecular plant-parasitic nematodes. Bioinformatics 36:1052- characterization of three species of the genus 1056. DOI: 10.1093/bioinformatics/btz707 Pratylenchoides Winslow, 1958 (Tylenchina, RASKI, D.J. 1975. Revision of the genus Paratylenchus Merliniidae, Pratylenchoidinae) from Iran. Journal of Micoletzky, 1922 and descriptions of new species. Crop Protection 3 (supplementary): 691-709. Part I of 3 parts. Journal of Nematology 7: 15-34. PENEVA, V., NEILSON, R., BOAG, B. & BROWN, D.J.F. REIS, C.S., DOS SANTOS, M.C.V., MARAIS, M., DE 2000. Criconematidae (Nematoda) from oak forests in SANTOS, M.S.N.A., DUYTS, H., FREITAS, H., VAN DER two nature reserves in Russia. Systematic Parasitology PUTTEN, W.H. & DE ABRANTES, I.M.O. 2010. First 46: 191-201. DOI: 10.1023/a:1006338019502 record of Helicotylenchus varicaudatus Yuen, 1964 POSADA, D. 2008. jModelTest: phylogenetic model (Nematoda: Hoplolaimidae) parasitizing Ammophila averaging. Molecular Biology and Evolution 25: arenaria (L.) Link in Portuguese coastal sand dunes. 1253-1256. DOI: 10.1093/molbev/msn083 Phytopathologia Mediterranea 49: 212-226. POWERS, T., HARRIS, T., HIGGINS, R., MULLIN, P. & RONQUIST, F. & HUELSENBECK, J.P. 2003. MrBayes 3: POWERS, K. 2017. An 18S rDNA perspective on the Bayesian phylogenetic inference under mixed models. classification of Criconematoidea. Journal of Bioinformatics 19: 1572-1574. Nematology 49: 236-244. DOI: 10.1093/bioinformatics/btg180 POWERS, T.O., HARRIS, T., HIGGINS, R., SUTTON, L. & RYBARCZYK-MYDŁOWSKA, K., DMOWSKA, E. & POWERS, K.S. 2010. Morphological and molecular KOWALEWSKA, K. 2019. Phylogenetic studies on characterization of Discocriconemella inarata, an three Helicotylenchus species based on 28S rDNA endemic nematode from North American native and mtCOI sequence data. Journal of Nematology 51: tallgrass prairies. Journal of Nematology 42: 35-45. 1-17. DOI: 10.21307/jofnem-2019-033 POWERS, T.O., BERNARD, E.C., HARRIS, T., HIGGINS, R., SEINHORST, J.W. 1991. The identity of Tylenchus OLSON, M., LODEMA, M., MULLIN, P., SUTTON, L. & robustus de Man, 1876, with remark on distortions in POWERS, K.S. 2014. COI haplotype groups in de Man’s (1876) figures. Nematologica 37: 119-122. Mesocriconema (Nematoda: Criconematidae) and DOI: 10.1163/187529291X00132 their morphospecies associations. Zootaxa 3827: 101- SIDDIQI, M.R. 2000. Tylenchida: Parasites of Plants and 146. DOI: 10.11646/zootaxa.3827.2.1 Insects. UK, CAB International. 833 pp. POWERS, T.O., BERNARD, E.C., HARRIS, T., HIGGINS, R., SINGH, P.R., NYIRAGATARE, A., JANSSEN, T., COUVREUR, OLSON, M., OLSON, S., LODEMA, M., MATCZYSZYN, J., M., DECRAEMER, W. & BERT, W. 2018. MULLIN, P., SUTTON, L. & POWERS, K.S. 2016A. Morphological and molecular characterisation of Species discovery and diversity in Lobocriconema Pratylenchus rwandae n. sp. (Tylenchida: (Criconematidae: Nematoda) and related plant-parasitic Pratylenchidae) associated with maize in Rwanda. nematodes from North American ecoregions. Zootaxa Nematology 20: 781-794. DOI: 10.1163/15685411- 4085: 301-344. DOI: 10.11646/zootaxa.4085.3.1 00003175 POWERS, T.O., MULLIN, P., HIGGINS, R., HARRIS, T. & STEEL, H., COOMANS, A., DECRAEMER, W., MOENS, T. & POWERS, K.S. 2016B. Description of Mesocriconema BERT, W. 2014. Nematodes from terrestrial, ericaceum n. sp. (Nematoda: Criconematidae) and freshwater and brackish water habitats in Belgium: an notes on other nematode species discovered in an updated list with special emphasis on compost ericaceous heath bald community in Great Smoky nematodes. Zootaxa 3765: 143-160. Mountains National Park, USA. Nematology 18: 879- DOI: 10.11646/zootaxa.3765.2.3 903. DOI: 10.1163/15685411-00003001 SUBBOTIN, S.A., WAEYENBERGE, L. & MOENS, M. 2000. QING, X., SLOS, D., CLAEYS, M. & BERT, W. 2017. Identification of cyst forming nematodes of the genus Ultrastructural, phylogenetic and rRNA secondary Heterodera (Nematoda: Heteroderidae) based on the structural analysis of a new nematode (Nematoda: ribosomal DNA-RFLPs. Nematology 2: 153-164. Tylenchomorpha) from mushroom with recovery of DOI: 10.1163/156854100509042 intestinal crystals. Zoologischer Anzeiger 269: 13-25. SUBBOTIN, S.A., STURHAN, D., RUMPENHORST, H.J. & DOI: 10.1016/j.jcz.2017.06.002 MOENS, M. 2003. Molecular and morphological
26 Molecular characterisation of plant-parasitic nematodes from Belgium
characterisation of the Heterodera avenae complex analysis tools. Nucleic Acids Research 25: 4876-4882. species (Tylenchida: Heteroderidae). Nematology 5: DOI: 10.1093/nar/25.24.4876 515-538. DOI: 10.1163/1568541033226832471 THORNE, G. 1949. On the classification of the SUBBOTIN, S.A., VOVLAS, N., CROZZOLI, R., STURHAN, Tylenchida, new order (Nematoda, Phasmidia). D., LAMBERTI, F., MOENS, M. & BALDWIN, J.G. 2005. Proceedings of Helminthological Society of Phylogeny of Criconematina Siddiqi, 1980 Washington 16: 37-73. (Nematoda: Tylenchida) based on morphology and TZORTZAKAKIS, E.A., ARCHIDONA-YUSTE, A., LIEBANAS, D2-D3 expansion segments of the 28S-rRNA gene G., BIRMPILIS, I. G., CANTALAPIEDRA-NAVARRETE, sequences with application of a secondary structure C., NAVAS-CORTES, J.A., CASTILLO, P. & model. Nematology 7: 927-944. PALOMARES-RIUS, J.E. 2016. Rotylenchus cretensis n. DOI: 10.1163/1568541057761863079 sp. and R. cypriensis Antoniou 1980. (Nematoda: SUBBOTIN, S.A., STURHAN, D., CHIZHOV, V.N., VOVLAS, Hoplolaimidae) recovered from the rhizosphere of N. & BALDWIN, J.G. 2006. Phylogenetic analysis of olive at Crete (Greece) with a molecular phylogeny of Tylenchida Thorne, 1949 as inferred from D2 and D3 the genus. European Journal of Plant Pathology 144: expansion fragments of the 28S rRNA gene 167-184. DOI: 10.1007/s10658-015-0760-2 sequences. Nematology 8: 455-474. VAN DEN BERG, E., TIEDT, L.R. & SUBBOTIN, S.A. 2012. DOI: 10.1163/156854106778493420 Morphological and molecular characterisation of SUBBOTIN, S.A., STURHAN, D., VOVLAS, N., CASTILLO, Criconemoides brevistylus Singh & Khera, 1976 and P., TAMBE, J.T., MOENS, M. & BALDWIN, J.G. 2007. C. obtusicaudatus Heyns, 1962 from South Africa Application of the secondary structure model of (Nematoda: Criconematidae) with first description of rRNA for phylogeny: D2-D3 expansion segments of a male C. obtusicaudatus and proposal of new the LSU gene of plant-parasitic nematodes from the synonyms. Nematology 14: 961-976. family Hoplolaimidae Filipjev, 1934. Molecular DOI: 10.1163/156854112X640304 Phylogenetics and Evolution 43: 881-890. VAN DEN BERG, E., YEATES, G.W., NAVAS-CORTÉS, J.A., DOI: 10.1016/j.ympev.2006.09.019 PLOEG, A.T., TIEDT, L.R., SUBBOTIN, S.A., ROBERTS, SUBBOTIN, S.A., CHITAMBAR, J.J., CHIZHOV, V.N., P.A. & COYNE, D.L. 2013. Morphological and STANLEY, J.D., INSERRA, R.N., DOUCET, M.E., molecular characterisation and diagnostics of some MCCLURE, M., YE, W., YEATES, G.W., MOLLOV, D.S., species of Scutellonema Andrássy, 1958 (Tylenchida: CANTALAPIEDRA-NAVARRETE, C., VOVLAS, N., VAN Hoplolaimidae) with a molecular phylogeny of the DEN BERG, E. & CASTILLO, P. 2014. Molecular genus. Nematology 15: 719-745. phylogeny, diagnostics, and diversity of plant-parasitic DOI: 10.1163/15685411-00002714 nematodes of the genus Hemicycliophora (Nematoda: VAN DEN BERG, E., TIEDT, L.R. & SUBBOTIN, S.A. 2014A. Hemicycliophoridae). Zoological Journal of the Morphological and molecular characterisation of Linnaean Society 171: 475-506. DOI: 10.1111/zoj.12145 several Paratylenchus Micoletzky, 1922 (Tylenchida: SUBBOTIN, S.A., VOVLAS, N., YEATES, G.W., Paratylenchidae) species from South Africa and USA, HALLMANN, J., KIEWNICK, S., CHIZHOV, V.N., together with some taxonomic notes. Nematology 16: MANZANILLA-LÓPEZ, R.H., INSERRA, R.N. & 323-358. DOI: 10.1163/15685411-00002769 CASTILLO, P. 2015. Morphological and molecular VAN DEN BERG, E., TIEDT, L.R., INSERRA, R.N., STANLEY, characterisation of Helicotylenchus pseudorobustus J.D., VOVLAS, N., PALOMARES-RIUS, J.E., CASTILLO, (Steiner, 1914) Golden, 1956 and related species P. & SUBBOTIN, S.A. 2014B. Morphological and (Tylenchida: Hoplolaimidae) with a phylogeny of the molecular characterisation of some genus. Nematology 17: 27-52. Hemicriconemoides species (Nematoda: SUBBOTIN, S.A., TOUMI, F., ELEKÇIOǦLU, I.H., Criconematidae) together with a phylogeny of the WAEYENBERGE, L. & TANHA MAAFI, Z. 2018. DNA genus. Nematology 16: 519-553. barcoding, phylogeny and phylogeography of the cyst DOI: 10.1163/15685411-00002786 nematode species of the Avenae group from the genus VAN DEN BERG, E., TIEDT, L.R., INSERRA, R.N., STANLEY, Heterodera (Tylenchida: Heteroderidae). Nematology J.D., VOVLAS, N., PALOMARES-RIUS, J.E., CASTILLO, 20: 671-702. DOI: 10.1163/15685411-00003170 P. & SUBBOTIN, S.A. 2015. Characterisation of a SWOFFORD, D.L. 2003. PAUP*: Phylogenetic Analysis topotype and other populations of Hemicriconemoides Using Parsimony (*and Other Methods). Version 4.0b strictathecatus Esser, 1960 (Nematoda: 10. USA, Sinauer Associates Inc. Criconematidae) from Florida with description of H. THOMPSON, J.D., GIBSON, T.J., PLEWNIAK, F., phoenicis sp. n. from the USA. Nematology 17: 265- JEANMOUGIN, F. & HIGGINS, D.G. 1997. The 300. DOI: 10.1163/15685411-00002866 CLUSTAL_X windows interface: flexible strategies VAN DEN BERG, E., TIEDT, L.R. & SUBBOTIN, S.A. 2017. for multiple sequence alignment aided by quality Morphological and molecular characterization of
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some Criconematidae (Nematoda, Tylenchida): Ogma data for H. ortonwilliamsi from Spain and H. wessoni decalineatus (Chitwood, 1957) Andrassy, 1979, from Florida. Nematology 8: 511-519. Criconema silvum (van den Berg, 1984) Raski & Luc, DOI: 10.1163/156854106778614047 1985 and Neobakernema variabile (Raski & Golden, VOVLAS, N., SUBBOTIN, S.A., TROCCOLI, A., LIÉBANAS, 1966) Ebsary. Russian Journal of Nematology 25: G. & CASTILLO, P. 2008. Molecular phylogeny of the 101-119. genus Rotylenchus (Nematoda, Tylenchida) and VAN DEN BERG, E., TIEDT, L.R., LIÉBANAS, G., description of a new species. Zoologica Scripta 37: CHITAMBAR, J.J., STANLEY, J.D., INSERRA, R.N., 521-537. DOI: 10.1111/j.1463-6409.2008.00337.x CASTILLO, P. & SUBBOTIN, S.A. 2018. Morphological WALLACE, H.R. & GREET, D.N. 1964. Observations on and molecular characterisation of two new the taxonomy and biology of Tylenchorhynchus Hemicycliophora species (Tylenchida: macrurus (Goodey, 1932) Filipjev, 1936 and Hemicycliophoridae) with a revision of the taxonomic Tylenchorhynchus icarus sp. nov. Parasitology 54: status of some known species and a phylogeny of the 129-144. genus. Nematology 20: 319-354. WHITEHEAD, A.G. & HEMMING, J.R. 1965. A comparison DOI: 10.1163/15685411-00003143 of some quantitative methods of extracting small VAN MEGEN, H., VAN DEN ELSEN, S., HOLTERMAN, M., vermiform nematodes from soil. Annales of Applied KARSSEN, G., MOOYMAN, P., BONGERS, T., Biology 55: 25-38. DOI: 10.1111/j.1744- HOLOVACHOV, O., BAKKER, J. & HELDER, J. 2009. A 7348.1965.tb07864.x phylogenetic tree of nematodes based on about 1200 YAO, H., SONG, J., LIU, C., LUO, K., HAN, J., LI, Y., PANG, full-length small subunit ribosomal DNA sequences. X., XU, H., ZHU, Y., XIAO, P. & CHEN, S. 2010. Use of Nematology 11: 927-950. ITS2 region as the universal DNA barcode for plants DOI: 10.1163/156854109X456862 and animals. PLoS ONE 5(10): e13102. VANDENBOSSCHE, B., VIAENE, N., DE SUTTER, N., MAES, DOI: 10.1371/journal.pone.0013102 M., KARSSEN, G. & BERT, W. 2011. Diversity and YEATES, G.W. & WOUTS, W.M. 1992. Helicotylenchus incidence of plant-parasitic nematodes in Belgian turf spp. (Nematoda: Tylenchida) from managed soils in grass. Nematology 13: 245-256. New Zealand. New Zealand Journal of Zoology 19: DOI: 10.1163/138855410X517084 13-23. DOI: 10.1080/03014223.1992.10423247 VOVLAS, N. 1980. Two new sheathoid nematodes YUEN, P.H. 1964. Four new species of Helicotylenchus (Nematoda: Criconematidae) from the Mediterranean Steiner (Hoplolaiminae: Tylenchida) and a region. Nematologia Mediterranea 8: 73-79. redescription of H. canadensis Waseem, 1961. VOVLAS, N., PALOMARES-RIUS, J.E., LIÉBANAS, G. & Nematologica 10: 373-387. CASTILLO, P. 2006. Re-establishment of DOI: 10.1163/187529264X00385 Hemicriconemoides promissus (Nematoda: URL: http://popart.otago.ac.nz/ (accessed: October 15, Criconematoidea) as a valid species, with additional 2019).
C.M. Etongwe, P.R. Singh, W. Bert and S.A. Subbotin. Молекулярная характеристика некоторых фитопаразитических нематод (Nematoda: Tylenchida) из Бельгии. Резюме. Используя морфологический, морфометрический и молекулярный анализы, одиннадцать валидных видов из девяти родов: Amplimerlinius icarus, Criconema annuliferum, Criconemoides informis, Helicotylenchus varicaudatus, Hemicriconemoides pseudobrachyurus, Hemicycliophora thienemanni, Mesocriconema xenoplax, Paratylenchus bukowinensis, P. nanus, Rotylenchus montanus and R. robustus и 12 неидентифицированных видов были обнаружены в пробах, собранных в девяти местах в Бельгии. Эти неопределенные образцы включали 6 видов Paratylenchus, один вид Helicotylenchus, три криконематиды и два вида Rotylenchus. В целом, 21 новых 18S рРНК, 69 28S рРНК, 10 ВТС рРНК and 51 COI генных последовательностей было получено для филогенетического анализа. Короткие описания, морфометрия и фотографии приводятся для некоторых видов. Основываясь на результатах молекулярного анализа, Hemicriconemoides promissus syn. n. синомизируется с H. pseudobrachyurus.
28 Molecular characterisation of plant-parasitic nematodes from Belgium
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