JOURNAL OF NEMATOLOGY Article | DOI: 10.21307/jofnem-2020-078 e2020-78 | Vol. 52

First report of hunaniense Wang & Wu, 1992 (Nematoda: ) in Vietnam

Huu Tien Nguyen1,2,3,*, Thi Duyen Nguyen1, 2, Thi Mai Linh Le1, 2 and Quang Phap Trinh1, 2,* Abstract 1Institute of Ecology and Biological For the first time, a survey of plant-parasitic in the Resources, Vietnam Academy Central Highlands of Vietnam discovered a population of Xiphinema of Sciences and Technology, hunaniense Wang & Wu, 1992. The Vietnamese population of 18 Hoang Quoc Viet, Cau Giay, X. hunaniense is characterized by having an offset lip region, lack 100000, Hanoi, Vietnam. of anterior genital branch, vagina directed backward, and a digitate tail. Morphological features and morphometrics of this population 2Graduate University of Science are in agreement with the type population of X. hunaniense except and Technology, Vietnam Academy for some variations. In addition, molecular characterization of this of Sciences and Technology, population and phylogenetic tree of 28S rDNA sequences of the 18 Hoang Quoc Viet, Cau Giay, genus are also provided. 100000, Hanoi, Vietnam.

3 Nematology Research Unit, Keywords Department of Biology, Ghent 28S rDNA, Dagger , Molecular identification, Plant-parasitic University, K.L. Ledeganckstraat nematodes, , Xiphinema hunaniense. 35, 9000, Ghent, Belgium. *E-mails: tien.quelampb@gmail. com, [email protected] This paper was edited by Zafar Ahmad Handoo. Received for publication May 26, 2020.

The genus Xiphinema Cobb, 1913, commonly known Nguyen, 2000), and thus, a higher diversity of as dagger nematodes, are migratory ectoparasitic Xiphinema spp. is expected in the country with nematodes that damage numerous wild and cultivated the use of molecular tools. Herein, a population of plants through direct feeding on the root and Xiphinema hunaniense Wang & Wu, 1992 in Vietnam transmission of plant (Taylor and Brown, 1997; is characterized by the combination of morphological Perry and Moens, 2013). This genus is distributed characters and molecular data. worldwide and is divided in two groups, group and non-Xiphinema americanum Material and methods group, with more than 260 valid species (Gutiérrez- Gutiérrez et al., 2012). The conserved morphology and Soil and root samples were collected from the upper overlapping morphometrics of some species groups 30 cm layer of forest soil in the Central Highlands of in the genus Xiphinema make quarantine regulations Vietnam. Nematodes were extracted and permanent and protection methods more difficult. Therefore, slides were made following Nguyen et al. (2019a). accurate identification of Xiphinema species using Pictures and measurements were recorded using integrate approach is strongly recommended to Carl Zeiss Axio Lab. A1 light microscope equipped create a basis for plant pest management. with a Zeiss Axiocam ERc5s digital camera. For In Vietnam, eight species of the genus Xiphinema molecular characterization, the 5′-end region of have been reported, however, molecular identification 28S rDNA was amplified using DP391/501 primers are not available for most of them (Nguyen and (5′-AGCGGAGGAAAAGAAACTAA-3′/5′-TCGG

© 2020 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/ Xiphinema hunaniense Wang & Wu, 1992 in Vietnam: Nguyen et al.

AAGGAACCAGCTACTA-3′) following Nguyen et al. (27-31) µm, a = 46 ± 2 (43-48), b = 5.8 ± 0.4 (5.4-6.1), (2019b). Forward and reverse sequences were assem­ c = 46 ± 4 (42-49), c′ = 1.6 ± 0.1 (1.5-1.7). bled and analyzed using Geneious R11 (Nguyen et al., 2019b, 2019c). The best fit model was chosen using Remarks Mega 7 following Nguyen et al. (2019b). The females of the Vietnamese population of Results and discussion X. hunaniense is characterized by an offset lip region from body contour, lack of anterior genital branch, vagina Measurements directed slightly backward, and a digitate tail (Fig. 1). Morphology and morphometrics of this population are Eight females: L = 2,095 ± 130 (1,947-2,189) µm, highly similar to the type population of X. hunaniense V% = 24.7 ± 0.4 (24.2-25), Odontostyle = 126 ± 5 (120-130) except for smaller a value (43-48 vs 51-57), c value (42- µm, Odontophore = 71 ± 1 (70-71) µm, Stylet = 196 ± 6 49 vs 53-63), longer stylet (190-201 µm vs 180-187 µm), (190-201) µm, Tail length = 45 ± 1 (44-46) µm, Lip wider width at pharyngo-intestinal junction (43-44 µm width = 13.6 ± 0.1 (13.5-13.7) µm, Lip height = 5.4 ± 0.2 vs 21-23 µm). However, these morphometric variations (5.2-5.6) µm, Pharynx = 359 ± 3 (356-361) µm, Anterior have been reported from other populations of end to guiding ring = 121 ± 7 (114-127) µm, Width at X. hunaniense (Luc, 1981; Wu et al., 2007; Long et al., pharyngo-intestinal junction = 44 ± 1 (43-44) µm, Width 2014). Two 28S rDNA sequences (1 bp difference) at mid-body = 46 ± 1 (45-47) µm, Width at anus = 29 ± 2 of the Vietnamese population of X. hunaniense were

Figure 1: Xiphinema hunaniense from Vietnam. A: entire body; B: Pharyngeal region; C: Vulva region; D: Tail region.

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100 Xiphinema italiae KX062677 100 Xiphinema italiae KX062675 55 Xiphinema italiae FJ713153 73 Xiphinema italiae KX2442912 Xiphinema meridianum KX062679 95 Xiphinema pyrenaicum GU725073 100 Xiphinema adenohystherum KX244897 73 100 Xiphinema adenohystherum KX244898 Xiphinema adenohystherum GU725075 Xiphinema hispidium KX244906 100 Xiphinema histriae MK801305 65 100 Xiphinema histriae MK801303 Xiphinema sphaerocephalum KX244917 Xiphinema herakliense KM586352 68 Xiphinema barense K199692 100 Xiphinema barense K199691 100 Xiphinema nuragicum GU725067 100 Xiphinema nuragicum GU725069 Xiphinema nuragicum GU725070 KX062673 Xiphinema conurum 84 100 Xiphinema oleae KU171038 Xiphinema oleae KU171037 76 Xiphinema sp. KX062686 100 Xiphinema sp. KX062687 Xiphinema turcicum KX244919 100 98 Xiphinema turcicum KX244918 100 Xiphinema lupini HM921352 Xiphinema lupini KC567183 100 Xiphinema lapidosum MK801307 Xiphinema elongatum KF430802 83 100 100 Xiphinema elongatum KF430803 Xiphinema elongatum E140790 100 Xiphinema elongatum KF430801 75 MF510430 Xiphinema insigne MT193456 92 100 Xiphinema insigne AY601619 Xiphinema insigne MT193455 100 Xiphinema radicicola AY601622 Xiphinema hangzhouense MF538772 100 Xiphinema setariae MF510429 99 93 Xiphinema chambersi KU680967 100 Xiphinema chambersi KT698208 97 Xiphinema chambersi KT698207 Xiphinema chambersi KU680965 100 Xiphinema chambersi AY601617 Xiphinema naturale DQ299515 93 Xiphinema hunaniense MT513135 Xiphinema hunaniense MT513136 Xiphinema hunaniense E188840 99 Xiphinema hunaniense KF290564 99 Xiphinema hunaniense MH156804 100 Xiphinema hunaniense KP793049 Xiphinema hunaniense EF026090 Xiphinema hunaniense E188841 Xiphinema hunaniense MF510432 73 Xiphinema hunaniense E188839 Xiphinema hunaniense KP793048 Xiphinema brasiliense AY601616 100 Xiphinema brasiliense KP793050 Xiphinema macrodora KU171044 100 Xiphinema macrodora KU171043 Xiphinema macrodora KU171042 56 Xiphinema macrodora KU171041 Xiphinema macrodora KU171040 100 Xiphinema cadavalense KX244900 Xiphinema baetica KC567168 100 Xiphinema andalusiense KX244887 100 Xiphinema andalusiense KX244885 Xiphinema andalusiense KX244886 62 100 Xiphinema coxi KC567179 Xiphinema coxi KC567174 100 Xiphinema coxi KC567177 Xiphinema belmontense KC567172 100 51 Xiphinema bakeri KF292278 Xiphinema bakeri KF292277 100 Xiphinema diversicaudatum JQ780360 100 Xiphinema diversicaudatum JQ780366 Xiphinema pseudocoxi KX244915 Xiphinema globosum GU549474 Xiphinema turdetanense KC567186 99 Xiphinema castilloi KF446655 56 Xiphinema sp. LT883657 99 Xiphinema montenegrinum MT271611 100 66 61 Xiphinema pyrenaicum AY601626 Xiphinema cretense KJ802879 100 Xiphinema cretense KJ802878 99 Xiphinema basiri AY601629 100 Xiphinema basiri AY601630 Xiphinema macroacanthum HF546081 95 HM921406 Xiphinema index HM921363 63 100 Xiphinema index HM921364 Xiphinema index AY584243 96 Xiphinema tica KY623485 Xiphinema tica KY623488 Xiphinema tica KY623492 100 Xiphinema tica KY623486 Xiphinema tica KY623489 99 Xiphinema americanum KU680975 99 Xiphinema americanum KU680974 KU680968 100 Xiphinema brevicollum H84473 96 Xiphinema brevicollum KR095280 macrosoma AY580055 Longidorus dunensis AY593057 0.050

Figure 2: BI phylogenetic tree generated from 28S rDNA sequences (GTR + G + I model, 1 × 106 generations, 20% Burn-in). Bayesian posterior probabilities (in percentage) are given next to each node. Sequences of X. hunaniense from Vietnam are in bold font.

3 Xiphinema hunaniense Wang & Wu, 1992 in Vietnam: Nguyen et al. obtained, 942 to 944 bp long. These sequences are Xiphinema americanum-group complex (Nematoda: 98.9 to 99.5% similar (3-8 bp difference) to 28S rDNA Longidoridae), as inferred from nuclear and mito­ sequences of X. hunaniense from other populations. chondrial DNA sequences and morphology. European The Bayesian inference phylogenetic tree showed that Journal of Plant Pathology 13 4:561– 97. 28S rDNA sequences of the Vietnamese population of Long, H., Ling, X., Li, Y. and Zheng, Y. 2014. X. hunaniense were placed together with sequences Molecular and morphological identification of of X. hunaniense from other populations (100% PP) Xiphinema hunaniense on the Juniperus chinensis and this group has a sister relationship (73% PP) to imported from Thailand. Plant Pathology & Quarantine 4:92–8. the sequences of X. brasiliense (Fig. 2). Loof, P. A. A., Luc, M. and Baujard, P. 1996. A revised When it comes to morphology, X. hunaniense polytomous key for the identification of species of the is closest to X. radicicola, and therefore, it has been genus Xiphinema Cobb, 1913 (Nematoda: Longidoridae) synonymized with X. radicicola by Loof et al. (1996). with exclusion of the X. americanum-group: Supplement However, based on the observation of different 2. Systematic Parasitology 33:23–9. populations of X. hunaniense and X. radicicola, Robbins Luc, M. 1981. Observations on some Xiphinema and Wang (1998) re-established X. hunaniense as a species with the female anterior genital branch valid species that was agreed by Zheng and Brown reduced or absent (Nematoda: Longidoridae). Revue (1999). 28S rDNA sequences of X. hunaniense has a de Nématologie 4:157–67. sister relationship to X. brasiliense, but X. hunaniense Nguyen, N. C. and Nguyen, V. T. 2000. Fauna of can be differentiated from X. brasiliense by the Vietnam, plant-parasitic nematodes Science and Tech­ moderately offset terminal peg vs distinct peg-shaped nics Publishing House, Hanoi. tail and X. brasiliense usually has more posterior Nguyen, H. T., Le, T. M. L., Nguyen, T. D., Liebanas, vulva position. Besides, the 28S rDNA sequences of G., Nguyen, T. A. D. and Trinh, Q. P. 2019a. Description of X. hunaniense from Vietnam were only 87 to 88% Geocenamus vietnamensis sp. n. (Nematoda: Merliniidae) similar (84-85 bp difference) to X. brasiliense. from Vietnam. Journal of Nematology 51:e2019–25. Due to the conserved morphology in some Nguyen, H. T., Phap, T. Q., Couvreur, M., Singh, Xiphinema species groups, i.e. X. hunaniense – P. R., Decraemer, W. and Bert, W. 2019b. Description X. radicicola – X. brasiliense (Zheng and Brown, 1999) of Rotylenchus rhomboides n. sp. and a Belgian population of Rotylenchus buxophilus (Tylenchomorpha: or X. americanum group (Gutiérrez-Gutiérrez et al., Hoplolaimidae). Journal of Nematology 51:1–20. 2012), the combination of morphological characters and Nguyen, H. T., Phap, T. Q., Couvreur, M., Singh, P. molecular data is needed to identify Xiphinema species. R., Decraemer, W. and Bert, W. 2019c. Molecular and This is the first report of X. hunaniense in Vietnam with morphological characterisation of a new root-lesion the support of molecular data of 28S rDNA sequences, nematode, Pratylenchus horti n. sp. (Tylenchomorpha: adding to the total number of nine Xiphinema species Pratylenchidae) from Ghent University Botanical Gar­ in Vietnam, including X. americanum, X. brasiliense, den. Nematology 21:739–52. X. brevicolle, X. diffusum, X. elongatum, X. insigne, Perry, R. and Moens, M. 2013. Plant nematology X. longicaudatum, X. radicicola, and X. hunaniense. CABI, London. Robbins, R. T. and Wang, S. 1998. A comparison Acknowledgments between Xiphinema radicicola and X. hunaniense. Program and Abstracts of the 24th International Sym­ This research was supported by a fund from the posium of the European Society of Nematologists, Institute of Ecology and Biological Resources (code: Dudee, Scotland, pp 100. IEBR ThST.8-20). Huu Tien Nguyen thanks to a special Taylor, C. A. and Brown, D. J. F. 1997. Nematode research fund from Ghent University (BOF-DOS vectors of plant viruses CABI, Wallingford. 01W02619) for his study in Belgium. The authors would Wu, Y., Zheng, J. and Robbins, R. T. 2007. Molecular characterization of a Xiphinema hunaniense like to thank Dr. Bui Van Thanh for his help in sampling. population with morphometric data of all four juvenile stages. Journal of nematology 39:37–42. Zheng, J. and Brown, D. J. F. 1999. Xiphinema References insigne Loos, 1949 and X. hunaniense Wang & Wu, 1992 from Hangzhou, China, and synonymization of X. savaryi Gutiérrez-Gutiérrez, C., Cantalapiedra-Navarrete, Lamberti, Troccoli and Agostinelli, 1997 with X. insigne, C., Decraemer, W., Vovlas, N., Prior, T., Palomares- and X. siamense Lamberti, Troccoli & Agrostinelli, 1997 Rius, J. and Castillo, P. 2012. Phylogeny, diversity, with X. radicicola Goodey, 1936 (Nematoda: Longi­ and species delimitation in some species of the doridae). Russian Journal of Nematology 7:127– 37.

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