JOURNAL OF NEMATOLOGY Article | DOI: 10.21307/jofnem-2019-023 e2019-23 | Vol. 51 Description of Rotylenchus rhomboides n. sp. and a Belgian population of Rotylenchus buxophilus (Tylenchomorpha: Hoplolaimidae) Huu Tien Nguyen1,2,3, Quang Phap Trinh1,2,*, Marjolein Couvreur3, Phougeishangbam Rolish Singh3, Abstract Wilfrida Decraemer3, and Wim Bert3 1Institute of Ecology and Biological During a survey in the Botanical garden of Ghent University, a new Resource, Vietnam Academy of species Rotylenchus rhomboides n. sp. and a population of Rotylen- Sciences and Technology, 18 chus buxophilus were found. Rotylenchus rhomboides n. sp. is char- Hoang Quoc Viet, Cau Giay, Hanoi, acterized by the presence of a rhomboid-like widening of the mid- Vietnam. ridge of lateral field at the level of vulva, a feature previously unknown 2Graduate University of Science within the genus. The population of the new species, composed only and Technology, Vietnam Academy by females, has a rounded labial region with 4 to 5 annuli, robust sty- of Sciences and Technology, let (31–37 μ m long), short dorsal esophageal gland (9–19 μ m) over- 18 Hoang Quoc Viet, Cau Giay, lap of the intestine, vulva located slightly posterior to mid-body, and 100000 Hanoi, Vietnam. hemispherical or rounded tail shape with large phasmids located 3 to 3Nematology Research Unit, 5 annuli anterior to the level of anus. The hierarchical cluster analysis Department of Biology, Ghent based on morphological features indicated that the new species University, K.L. Ledeganckstraat closely resembles R. corsicus, R. gracilidens, and R. rugatocuticu- 35, 9000 Ghent, Belgium. latus. The DNA analyses of the D2-D3 of 28S rDNA, ITS rDNA, and *E-mail: [email protected] COI mtDNA sequences of Rotylenchus rhomboides n. sp. show a close relationship with R. buxophilus, R. goodeyi, R. laurentinus, This paper was edited by Zafar R. pumilus, and R. incultus, all of which can also be differentiated Ahmad Handoo. from the new species by morphological features. The combination Received for publication December of morphological, morphometric, and molecular characteristics con- 18, 2018. firmed the new species and the first report ofR. buxophilus on yam (Dioscorea tokoro) in Belgium. Key words 28S–D2D3, Hierarchical cluster, COI, Dioscorea tokoro, ITS, Mor- phology, Musa basjoo, Phylogeny, Plant-parasitic, Rotylenchus, Systematic, Taxonomy. The type species Rotylenchus robustus (De man, Vovlas, 2005; Vovlas et al., 2008; Atighi et al., 2011; 1876) was originally described as Tylenchus robustus Cantalapiedra-Navarrete et al., 2012, 2013; Atighi by De man (1876). In a publication concerning free-liv- et al., 2014; Aliramaji et al., 2015; Noruzi et al., 2015; ing nematodes and their relation to the parasitic nem- Talezari et al., 2015; Golhasan et al., 2016). According atodes, Filipjev (1934) proposed the new genus Rot- to Castillo and Vovlas (2005), the number of Rotylen- ylenchus based on the type species T. robustus (De chus species is second highest in Europe, after Asia. man, 1876). Several species of Rotylenchus are of economic im- The genus Rotylenchus is widely distributed all portance in agriculture, among them, only R. robustus, over the world and has been recorded from all con- R. buxophilus (Golden, 1956), R. uniformis (Thorne, tinents with 103 valid species to date (Castillo and 1949; Loof and Oostenbrink, 1958), and R. goodeyi © 2019 Authors. This is an Open Access article licensed under the Creative 1 Commons CC BY 4.0 license, https://creativecommons.org/licenses/by/4.0/ Description of Rotylenchus rhomboides n. sp. and a Belgian population of Rotylenchus buxophilus (Loof and Oostenbrink, 1958) have been reported in Molecular characterization Belgium (Steel et al., 2014; Viaene et al., 2017). They are the main cause of yield losses in many agricultural crops Digital light microscope pictures were taken from such as carrot, olive, orange, mango, soybean, broc- temporary slides as morphological vouchers. Then, coli, cabbage, tomatoes, etc. Rotylenchus robustus the nematodes were cut into pieces and put in the is considered as the most common species worldwide Eppendorf tubes with 20 µl of WLB (50 mM KCl; and has been reported in 25 countries and islands on 10 mM Tris pH 8.3; 2.5 mM MgCl2; 0.45% NP 40 all continents except Antarctica. Rotylenchus buxo- (Tergitol Sigma); 0.45% Tween 20) and were fro- philus is also a widely distributed species, occurring in zen for at least 10 min at −20°C. One μ l proteinase Europe, Asia, North America, and New Zealand. Rot- K (1.2 mg ml−1) was added before the incubation in ylenchus uniformis was found in the Netherlands and a PCR machine for 1 hr at 65°C and 10 min at 95°C New Zealand. Rotylenchus goodeyi was recorded from and centrifugation for 1 min at 14,000 rpm. Finally, the several localities in the UK, the Netherlands, Belgium, samples were stored at −20°C before running PCR Bulgaria, Slovakia, Spain, Switzerland, Luxemburg, (Singh et al., 2018). and Uzbekistan (Castillo and Vovlas, 2005). The 5′-end of the 28S rDNA region was amplified Herein, the new species Rotylenchus rhomboides using the primers DP391/501 (Nadler et al., 2006) n. sp. is characterized and a population of R. buxo- with the PCR reaction started at 94°C for 4 min, fol- philus is reported for the first time on yam Dioscorea( lowed by 5 cycles of 94°C for 30 s, 45°C for 30 s, and tokoro Makino). Both species were described based 72°C for 2 min. This step was followed by 35 cycles on morphology and morphometrics along with mo- of 94°C for 30 s, 54°C for 30 s, and 72°C for 1 min lecular characteristics and phylogeny of the D2-D3 and finished at 12°C for 10 min. For ITS rDNA region, expansion segment of 28S rDNA, ITS rDNA, and COI the primers Vrain2F/Vrain2R (Vrain et al., 1992) were mtDNA sequences. used with the PCR reaction started at 94°C for 4 min, followed by 50 cycles of 94°C for 30 s, 54°C for 30 s, and 72°C for 2 min. The cytochrome c oxidase subu- Materials and methods nit 1 (COI mtDNA) gene was amplified using the prim- ers JB3/JB4 according to the protocol of Derycke et Sampling and nematode extraction al. (2010). The successful PCR reactions were puri- fied and sequenced commercially by Macrogen Eu- The soil and root samples were collected around rope (Amsterdam, Nederland). the rhizosphere of banana (Musa basjoo Siebold & The forward and backward sequences were as- Zucc. ex Iinuma) (GPS coordinates N: 51o2′6.8′′, E: sembled in Geneious R11 (www.geneious.com) to get 3o43′22.7′′) and Yam (Dioscorea tokoro) (GPS coor- the consensus sequences. All the contigs were used dinates: N: 51o2′6.9′′, E: 3o43′22.6′′) at the Botanical for the BLAST search on GenBank to check for the garden of Ghent University. The nematodes were ex- closely related species (Altschul et al., 1997). Multiple tracted from soil and roots by the modified Baermann alignments of the different sequences of each gene tray method (Whitehead and Hemming, 1965). were made using MUSCLE in MEGA 7 (Kumar et al., 2016). The phylogenetic trees were created by using Morphological characterization MrBayes 3.2.6 (Huelsenbeck and Ronquist, 2001) Add-in in Geneious R11 (www.geneious.com) under Nematodes were fixed in Trump’s fixative (2% paraform- the nucleotide substitution models that were select- aldehyde + 2.5% glutaraldehyde in a 0.1 M Sorenson ed by using MEGA 7 (Sharma et al., 2012) based on buffer (Sodium phosphate buffer at pH 7.3)), then de- the BIC criterion. The selected models were HKY + G hydrated for mounting in glycerin on permanent slides for the D2-D3 of 28S rDNA and ITS rDNA sequenc- following the method described by Singh et al. (2018). es, and GTR + G + I for COI mtDNA sequences. The Microphotographs and drawings were made from Markov chains were set with 1 × 106 generations, 4 permanent slides by using an Olympus BX51 DIC Mi- runs, 20% burn-in, and subsampling frequency was croscope equipped with a drawing tube and digital 500 generations (Huelsenbeck and Ronquist, 2001). camera. Measurements were obtained based on light For D2-D3 of 28S rDNA data set, Helicotylenchus di- microscopic pictures using the software ImageJ 1.51. hystera (accession number: AB933471) and Helicoty- Illustrations were made by Illustrator ® CS 3 based lenchus multicinctus (accession number: MF401446) on pencil drawings and SEM pictures. For scanning were chosen as outgroups. The outgroups for ITS electron microscopy, specimens were processed and rDNA data set were Hoplolaimus columbus (acces- viewed following the procedure of Steel et al. (2011). sion number: FJ485623) and Hoplolaimus seinhorsti 2 JOURNAL OF NEMATOLOGY (accession number: KX446971), and Scutellone- In order to facilitate the identification of Rotylenchus ma brachyurus (accession number: JX472089) spp., based on a cluster analyses a web-based key and Scutellonema truncatum (accession number: was developed. The domain for this web-based key KX959308) were selected for COI mtDNA data set. was registered from the website: www.awardspace. com. The interface of this web-based key was written Cluster analysis and web-based key using Notepad++ v7.5.6 and the algorithm was based on Bray–Curtis similarity measure. The web-based key The new species was compared with 103 described can be freely accessed at http://nematodeidentifica- species, based on the tabular key of Castillo and Vo- tion.mypressonline.com/category/identification-tool/. vlas (2005). This was done using Hierarchical Cluster analysis implemented in the software Primer 6 (Clarke Results and Gorley, 2006) using Bray–Curtis similarity meas- ure with the percent similarity between species de- SYSTEMATICS fined by the average of the multiple characters.