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Molecular Characterisation of Some Plant-Parasitic Nematodes

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Molecular Characterisation of Some Plant-Parasitic Nematodes 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 2, 3 Sergei A. Subbotin 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
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