CC BY
18Russian Journal of Nematology, 2019, 27 (1), 29 - 36
Report of Neomisticius rhizomorphoides (Rühm, 1955) Siddiqi, 1986 (Tylenchida: Anguinidae) from a cherry tree in California, USA
Sergei A. Subbotin1' 2 and Vladimir N. Chizhov2
'Plant Pest Diagnostic Centre, California Department of Food and Agriculture, 3294 Meadowview Road, 95832-1448, Sacramento, CA, USA 2Centre of Parasitology, A.N. Severtsov Institute of Ecology and Evolution, Russian Academy of Sciences,
Leninskii Prospect 33, 117071, Moscow, Russia e-mail: sergei.a. [email protected]
Accepted for publication 2 August 2019
Summary. Neomisticius rhizomorphoides was reported from a cherry tree in California, USA. Free-living adult and juvenile nematodes were found in tunnels made by bark beetles Xeleborus dispar and Xyleborinus saxeseni. The tunnels were infested by two species of ascomycete fungi from the genus Ophiostoma. Morphological and molecular characterisations of free-living adult stages of N. rhizomorphoides are given. Phylogenetic analysis of partial 28S rRNA gene sequences revealed that N. rhizomorphoides belongs to the family Anguinidae.
Key words: Ophiostoma, Paurodontidae, phylogeny, Xeleborus dispar, Xyleborinus saxeseni.
In July 2018, several branches of a cherry tree collected in Trinity County, California, USA and infected by bark beetles were received by the Plant Pest Diagnostic Centre of California Department of Food and Agriculture, Sacramento. Analysis of this wood sample revealed the presence of numerous nematodes inhabiting bark tunnels. Based on morphology this nematode was identified as Neomisticius rhizomorphoides (Rühm, 1955) Siddiqi, 1986. The genus Neomisticius belongs to the subfamily Paurodontinae Thorne, 1941, family Paurodontidae Thorne, 1941, superfamily Sphaerularioidea Lubbock, 1861 (Poinar, 1975), suborder Hexatylina Siddiqi, 1980 of the order Tylenchida Thorne, 1949 according to Siddiqi (2000). The genus Neomisticius contains only one species. The present study provides a short morphological and molecular characterisation of free-living adult stages of N. rhizomorphoides and a position of this species within the order Tylenchida based on the partial 28S rRNA gene sequences.
MATERIAL AND METHODS
Nematode sample. Wood samples were collected from a cherry tree (Prunus sp.) growing in an orchard in Junction City, Trinity County, California, USA. Nematodes were extracted by
placing small pieces of tree branches into a Petri dish with distilled water. Measurements and light micrographs were taken of live specimens with an automatic Infinity 2 camera attached to a compound Olympus BX51 microscope equipped with Nomarski interference contrast.
DNA extraction, PCR, sequencing and phylogenetic analysis. DNA was extracted from several specimens using the proteinase K protocol. DNA extraction, PCR and cloning protocols were used as described by Tanha Maafi et al. (2003). The following primer set was used for PCR: the forward D2A (5'-ACA AGT ACC GTG AGG GAA AGT TG-3') and the reverse D3B (5'-TCG GAA GGA ACC AGC TAC TA-3') primers (Subbotin et al., 2006) for amplification of the D2-D3 expansion segments of 28S rRNA gene and the forward TW81 (5'-GTT TCC GTA GGT GAA CCT GC-3') and the reverse AB28 (5'-ATA TGC TTA AGT TCA GCG GGT-3') primer (Tanha Maafi et al., 2003) for amplification of the ITS1-5.8-ITS2 rRNA gene. The D2-D3 of 28S rRNA PCR product was purified using QIAquick PCR purification kit (Qiagen, USA) and submitted for direct sequencing, whereas the ITS rRNA PCR product was purified using QIAquick Gel extraction kit (Qiagen), cloned into the pGEM-T vector and transformed into JM109 High Efficiency Competent Cells (Promega, USA).
© Russian Society of Nematologists, 2019; doi:10.24411/0869-6918-2019-10004
Fig. 1. A & D: Bark tunnels in cherry wood; B & C: Nematodes on wood surface. Arrows indicate nematode specimens. Scale bars: A & D - 1 mm; B & C - 100 ^m.
The ITS clone was submitted to PCR with same primers and then sequenced. Sequencing was conducted at Quintara Biosciences (CA, USA). The newly obtained sequences were submitted to the GenBank database under accession numbers: MK929283 (D2-D3 expansion fragments of 28S rRNA) and MK929281 (ITS rRNA).
The new sequence of the D2-D3 of 28S rRNA gene was aligned using ClustalX 1.83 (Thompson et al., 1997) (multiple alignment parameters: gap opening - 5 and gap extension - 3) with published gene sequences of related genera (Chizhov et al., 2012; Yaghoubi et al., 2014, 2018; Esmaeili et al., 2017, 2018; Mobasseri et al., 2017). Outgroup taxa for each dataset were chosen based on previously published data (Subbotin et al., 2006). Gblock version 0.91b was used to eliminate poorly aligned positions and divergent regions of an alignment (Talavera & Castresana, 2007). Sequence alignment was analysed with Bayesian inference (BI) using MrBayes 3.1.2 (Ronquist & Huelsenbeck, 2003) under the GTR + G + I model. BI analysis for each gene was initiated with a random starting tree and was run with four chains for 1.0 * 106 generations. Two runs were performed for each analysis. The Markov chains were sampled at intervals of 100 generations. After discarding burn-in samples (10%), a 50% majority rule consensus tree was generated. Posterior probabilities (PP) in percentage are given on appropriate clades.
RESULTS AND DISCUSSION
Neomisticius rhizomorphoides (Rühm, 1955) Siddiqi, 1986 (Figs 1-3)
Free-living females (n = 4): L = 1431.8 ± 231.8 (1137.5-1670) pm; maximum body width = 25.6 ± 2.4 (22.5-27.5) pm; a = 56.2 ± 10.6 (45.5-68.9); b = 7.9 ± 4.6 (4.9-10.3); c = 50.7 ± 0.9 (49.8-51.6); V = 90.8 ± 1.2 (89.4-91.9)%; stylet = 11.7 ± 0.9 (10.612.5) pm; tail = 26.6 ± 3.8 (22.5-30) pm.
Free-living males (n = 6): L = 976.3 ± 135.5 (770-1137.5) pm; maximum body width = 24.4 ±
1.0 (23.8-26.3) pm; a = 40.0 ± 5.1 (32.4-47.9); b =
8.1 ± 1.0 (6.7-9.3); c = 32.4 ± 5.7 (28-43.3); stylet = 12.5 ± 0.4 (11.9-13.1) pm; tail = 30.4 ± 3.5 (26.335) pm; spicules = 21.3 ± 1.6 (18.8-22.5) pm; gubernaculum = 7.6 ± 0.3 (7.5-8.1) pm.
Free-living female. Body long. Cuticle finely annulated. Lip region low and smooth. Stylet with small knobs. Orifice of dorsal pharyngeal gland to very close to stylet knobs. Procorpus cylindroid, metacorpus weakly developed, isthmus narrow and basal bulb large. No basal bulb extension projecting observed into lumen of intestine. Excretory pore near base of stylet. Postvulval uterine sac large. Female tail subcylindrical sometimes with a short terminal process.
Fig. 2. Neomisticius rhizomorphoides. Adults. A: Female and male, whole bodies; B-D: Anterior region of females; F, G, H, I: Posterior region of females, E: Lateral field of female. Arrow indicates excretory pore. Scale bars: 20 ^m.
Fig. 3. Neomisticius rhizomorphoides. Male and male juvenile. A & B: Anterior region of males; C: Anterior region of male juvenile; D: Lateral field of male; E-G: Posterior region of males. Arrow indicates excretory pore. Scale bars: 20 ^m.
Anguinidae
100
79
100
100
99
100
100
99
100
100
unidentified entomoparasitic nematode (DQ328725)
- Orrina phyllobia (KT192617)
- Neomisticius rhizomorphoides (MK929283, CD2767)
- Ficotylus congestae (EU018047)
- Ficotylus laselvae (MG835408)
— unidentified tylenchid nematode (KY750848) -Ditylenchus halictus (AY589364)
100
Ditylenchus destructor (FJ707365)
79 100
fc
— Nothotylenchus persicus (KT149799) Anguina agrostis (MH374272)
— Subanguina chilensis (DQ328724)
91
100
Ditylenchus dipsaci (JF327763) 1.— Ditylenchus drepanocercus (JQ429773) ~L Zeatylenchus pittosporum (KY067443) - Ditylenchus gallaeformis (KF494346)
Parasitylenchus sp. (DQ 328729)
100
100
100
100
100
rC
C,
100
98
Anguillonema iranicum (MG857082) Howardula phyllotretae (DQ328728) Anguillonema amolensis (MF134424)
- Bradynema listronoti (DQ915804)
- Allantonema mirable (JX291132)
100
C
-unidentified nematode (JX291133)
Rubzovinema sp. (KF155281) - Deladenus siricidicola (AY633444) Fergusobia camaldulensae (AY589346) -Skarbilovinema lyoni (DQ328733)
Howardula aoronymphium (AY589395)
99j- i 70 fl—
99
93
99
Sphaerularia bombi (DQ328726) Sphaerularia vespae (AB300596)
JL
— Paurodontoides siddiqii (MG836264) Sphaerularia sp. (MH590284)
— Veleshkinema iranicum (KM401545)
— Paurodontella iranica (KP642168)
-Abursanema iranicum (KF885742)
-Paurodontella gilanica (MF543010)
-Paurodontella persica (KP000034)
- Acrobeloides camberenensis (AF147069)
- Zeldia punctate (EU195988) 0.1
Fig. 4. Phylogenetic relationships within Anguinidae and Hexatylina. Bayesian 50% majority rule consensus tree from two runs as inferred from analysis of the D2-D3 of 28S rRNA gene sequence alignment under the GTR + I + G model. Posterior probabilities equal or more than 70% are given for appropriate clades. Original sequence of
Neomisticius rhizomorphoides is indicated by bold font.
Free-living males. Body shorter than in female. General morphology similar to female. Excretory pore near base of stylet. Lateral field with two lines. Testis single, anteriorly outstretched. Spicules paired, ventrally arcuate. Gubernaculum simple, arcuate. Bursa large and covering the entire tail.
Location and habit. Nematode adults and juveniles were found in bark tunnels (Fig. 3) of cherry tree collected in Trinity County, California, USA. The tunnels made by bark beetles Xeleborus dispar (Fabricius 1792) and Xyleborinus saxeseni (Ratzeburg 1837) (family Curculionidae) (Dr Alexey K. Tishechkin, pers. comm.) were infested by two species of ascomycete fungi from the genus Ophiostoma (Dr Suzanne Rooney Latham, pers. comm.).
Molecular characterisation and phylogenetic position. Phylogenetic position of N. rhizomorphoides within the Tylenchida obtained from the analysis of the D2-D3 of 28S rRNA gene sequence using the BI is given in Fig. 4. In the partial 28 S rRNA gene phylogenetic tree, N. rhizomorphoides was nested within Anguinidae and it was a sister taxon to Orrina phyllobia and an unidentified entomoparasitic nematode, although the statistical support for such relationships was low. BLAST search of the ITS rRNA gene sequence of N. rhizomorphoides revealed highest similarity with those of Orrina phyllobia (similarity - 79.2%, coverage - 42%) and Ditylenchus persicus (79.9% and 39%).
Neomisticius rhizomorphoides was described by Rühm (1955) in Germany as representative of the genus Anguillonema, A. rhizomorphoides. Different stages of this nematode were discovery from the body cavity and genitalia of the beetle Xyleborus dryographus (Ratzeburg, 1837) (family Curculionidae) and from bark tunnels, which were infested with fungal mycelium. Nematode eggs and other stages have been found in large numbers in layers of fungal mycelium (Sumenkova, 1975). The genus Anguillonema was poorly described by Fuchs (1938) and never correctly re-described in spite of the works of Rühm (1956) and Massey (1974). Golden (1971), Sumenkova (1975) and Andrassy (1976) placed this genus in Misticiinae (Paurodontidae) (Fortuner & Raski, 1987). Siddiqi (1986) erected a new genus Neomisticius for A. rhizomorphoides. He described the genus with stemlike basal extension. The base of the pharynx of A. rhizomorphoides was not described in the original publication by Rühm (1955) (Fortuner & Raski, 1987). Neomisticius differed from Misticius in a having different tail shape and bursa enveloping entire tail. Fortuner and Raski (1987) noticed that N. rhizonzorphoides was very similar to
Sychnotylenchus (Anguinidae) by anterior position of excretory pore, cylindroid female tail with rounded end, caudal alae enveloping male tail, and to Stictylus (synonym of Prothallonema, Sphaerulariidae sensu Siddiqi, 2000) by the corpus of the pharynx but without a stem at the posterior end of the glands. These authors considered N. rhizomorphoides as species inquirendum.
In the family Paurodontidae Siddiqi (2000) included seven genera: Paurodontus Thorne, 1941, Bealius Massey & Hinds, 1970, Luella Massey, 1974, Misticius Massey, 1967, Neomisticius, Paurodontella Husain & Khan, 1968 and Paurodontoides Jairajpuri & Siddiqi, 1969. Siddiqi (2000) questioned the validity of Paurodontidae and believed that most probably this family was junior synonym of Sphaerulariidae, since the type genus and other genera included in it were morphologically similar and were suspected of having similar life cycles to members of the latter group. Andrassy (2007) agreed and placed Neomisticius and other genera in family Sphaerulariidae Lubbock, 1861 and subfamily Paurodontinae Thorne, 1941. Recent phylogenetic analysis studies using 18S and partial 28S rRNA gene sequences confirmed that some species of Paurodontella (Yaghoubi et al., 2018) clustered with representatives of Sphaerulariidae; however, the positions of other species are still uncertain (Esmaeili et al., 2017). Thus, the revision of some genera of this family is still needed. It should be also noticed that the name of Paurodontidae was already used by Marsh (1887) for a family of Late Jurassic to Early Cretaceous mammals in the order Dryolestida.
ACKNOWLEDGEMENTS
Authors thank Dr Suzanne Rooney Latham (CDFA, CA, USA) for identification of fungi and Dr Alexey Tishechkin (CDFA, CA, USA) for identification of beetles.
REFERENCES
Andrassy, I. 1976. Evolution as a Basis for the Systematization of Nematodes. UK, Pitman Publishing
Ltd. 288 pp.
AndrAssy, I. 2007. Free-Living Nematodes of Hungary (Nematoda errantia), II. Pedozoologica Hungarica, no. 4 (C. Csuzdi & S. Mahunka Eds). Hungary, Hungarian Natural History Museum. 496 pp. Chizhov, V.N., Butorina, N.N. & Subbotin, S.A. 2012. Entomoparasitic nematodes of the genus Skarbilovinema: S. laumondi and S. lyoni (Nematoda:
Tylenchida), parasites of the flies of the family Syrphidae (Diptera) with phylogeny of the suborder Hexatylina. Russian Journal of Nematology 20: 141-155. Esmaeili, M., Heydari, R. & Ye, W. 2017. Molecular and morphological characterisation of Paurodontella persica n. sp. (Hexatylina: Sphaerulariidae) from soil in Iran. Nematology 19: 57-68. DOI: 10.1163/15685411-00003031 Esmaeili, M., Golhasan, B., Ye, W. & Heydari, R. 2018. Molecular and morphological characterization of Paurodontoides siddiqii n. sp. (Nematoda: Hexatylina, Sphaerulariidae) associated with bark samples of Pinus eldarica from western iran. Journal of Helminthology 29: 1-8. DOI: 10.1017/S0022149X1800103 Fortuner, R. & Raski, D.J. 1987. A review of Neotylenchoidea Thorne, 1941 (Nemata: Tylenchida). Revue de Nematologie 10: 257-267. Fuchs, A.G. 1938. Neue Parasiten und Halbparasiten bei Borkenkäfer und einige andere Nematoden (Teil II, III und IV). Zoologischer Jahresbericht 71: 123-190. Golden, A.M. 1971. Classification of the genera and higher categories of the order Tylenchida (Nematoda). In: Plant Parasitic Nematodes. Volume 1: Morphology, Anatomy, Taxonomy and Ecology (B.M. Zuckerman, W.F. Mai & R.A. Rohde Eds). pp. 191232. New York, USA, Academic Press. Marsh, O.C. 1887. American Jurassic mammals.
American Journal of Science 33: 327-348. Massey, C.L. 1974. Nematodes associated with tree-infesting insects Paurodontidae new family and Misticiinae new subfamily with a description of one new genus and four new species. Canadian Journal of Zoology 45: 779-786. DOI: 10.1139/z67-089 Mobasseri, M., Pedram, M. & Pourjam, E. 2017. A new species of the rare genus Anguillonema Fuchs, 1938 (Nematoda: Hexatylina, Sphaerularioidea) with its molecular phylogenetic study. Journal of Nematology 49: 286-294. Rühm, W. 1955. Über einige an holzbrütende Ipiden gebundene Nematodenarten. Zoologischer Anzeiger 155: 70-83.
Rühm, W. 1956. Die Nematoden der Ipiden.
Parasitologische Schriftenreihe 6: 1-437.
Ronquist, F. & Huelsenbeck, J.P. 2003. MrBayes 3: Bayesian phylogenetic inference under mixed models. Bioinformatics 19: 1572-1574. DOI: 10.1093/bioinformatics/btg180 Siddiqi, M.R. 1986. Tylenchida: Parasites of Plants and Insects. UK, Commonwealth Agricultural Bureaux. 645 pp. Siddiqi, M.R. 2000. Tylenchida: Parasites of Plants and
Insects. UK, CAB International. 833 pp. Sumenkova, N.I. 1975. [Nematodes of Plants and Soil. Neotylenchoidea.] USSR, Nauka. 200 pp. (in Russian).
Subbotin, S.A., Sturhan, D., Chizhov, V.N., Vovlas, N. & Baldwin, J.G. 2006. Phylogenetic analysis of Tylenchida Thorne, 1949 as inferred from D2 and D3 expansion fragments of the 28S rRNA gene sequences. Nematology 8: 455-474. DOI: 10.1163/156854106778493420 Talavera, G. & Castresana, J. 2007. Improvement of phylogenies after removing divergent and ambiguously aligned blocks from protein sequence alignments. Systematic Biology 56: 564-577. Tanha Maafi, Z., Subbotin, S.A. & Moens, M. 2003. Molecular identification of cyst-forming nematodes (Heteroderidae) from Iran and a phylogeny based on ITS-rDNA sequences. Nematology 5: 99-111. DOI: 10.1163/156854102765216731 Thompson, J.D., Gibson, T.J., Plewniak, F., Jeanmougin, F. & Higgins, D.G. 1997. The Clustal_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 24: 4876-4882. DOI: 10.1093/nar/25.24.4876 Yaghoubi, A., Pourjam, E. & Pedram, M. 2018. Paurodontella gilanica n. sp. (Nematoda: Sphaerularioidea) from Iran. Nematology 20: 471482. DOI: 10.1163/15685411-00003152 Yaghoubi, A., Pourjam, E., Pedram, M., Siddiqi, M.R. & Atighi, M.R. 2014. Description of Abursanema iranicum n. gen., n. sp. (Nematoda: Hexatylina, Sphaerularioidea) from Iran and its phylogenetic relationships. Zootaxa 3826: 301-314. DOI: 10.11646/zootaxa.3826.2.1.
С.А. Субботин и В.Н. Чижов. Сообщение о Neomisticius rhizomorphoides (Rühm, 1955) Siddiqi, 1986 (Tylenchida: Anguinidae) из вишневого дерева в Калифорнии, США.
Резюме. Neomisticius rhizomorphoides был выделен из вишневого дерева в Калифорнии, США. Свободноживущие взрослые стадии и личинки нематоды были обнаружены в туннелях, сделанных короедами: Xeleborus dispar и Xyleborinus saxeseni. Туннели были заражены двумя видами грибов аскомицетов из рода Ophiostoma. Даны морфологические и молекулярные характеристики свободноживущих взрослых стадий этой нематоды. Филогенетический анализ последовательностей гена 28S рРНК показал, что N. rhizomorphoides принадлежит к семейству Anguinidae.
