The genus Tylenchorhynchus Cobb, 1913 consists of a large number of migratory ecto-parasitic nematodes, commonly known as stunt nematodes, which are able to infect and cause considerable damage to the roots of a wide range of agricultural crops, including important food crops such as rice, corn and wheat (Decraemer and Geraert 2013; Abdulsalam et al. 2022). In an analysis of the plant-parasitic nematodes (PPNs) found in 16 provinces in Indonesia, Chaerani (2022) recently reported the occurrence of Tylenchorhynchus spp. on ten crop species. None of these Tylenchorhynchus populations had been identified to species level. This is unfortunate since the accurate identification of PPNs to species level is absolutely necessary to choose a proper management practice and, eventually, prevent the spread of highly pathogenic species (Hassan et al. 2013).

During an investigation of the presence of PPNs on some food crops in Central Java, Indonesia, rhizosphere soil samples were collected from paddy rice in a research field of the Agro-Technology Innovation Centre, Universitas Gadjah Mada, Berbah, Yogyakarta, and in a corn field in Klaten, Central Java. Nematodes were extracted from the soil using the modified Whitehead tray method (Southey 1986), fixed with formal acetic (FA; Hooper et al. 2005; except those specimens used for molecular analyses) and mounted on glass slides as semi-permanent specimen (nematodes were killed in hot distilled water and then transferred to fixative solution). Identification of the populations to species level was based on Handoo (2000). Morphometrics and morphological characters were measured and observed under an optical microscope.

Female body (n = 7; corn population) 702.4 ± 76.6 (559 to 772) µm long, arcuate ventrally to straight after fixation (Fig. 1a, d). a = 29.6 ± 3.4 (25.6 to 34.9), b = 4.6 ± 0.4 (3.9 to 5), c = 13.2 ± 1.9 (9.9 to 15.5), c’ = 3.1 ± 0.4 (2.4 to 3.6). Lip region slightly offset (Fig. 1b, e, i), 7.1 ± 0.5 (6.4 to 7.6) µm wide, 3.5 ± 0.3 (2.9 to 3.8) µm high, bearing either 2 or 3 annules. Stylet 19.8 ± 1.2 (18.7 to 21.7) µm long, knobs rounded at the basis and anterior surfaces slightly flat to convex. (Fig. 1b, e, i). Dorsal gland orifice (DGO) 2.8 ± 0.4 (2.3 to 3.3) µm posterior to anterior head end. Median bulb moderately developed (Fig. 1b, e), oval, occupying 6 to 7 body annules. Basal pharyngeal bulb elongated, not overlapping intestine. Lateral field with four incisures (Fig. 1g). Vulva body width 23.9 ± 2.2 (20.1 to 26.1) µm. Vulva slightly post-median (Fig. 1a, d). V% = 56.5 ± 1.3 (54.6 to 58.5). Two equally developed genital tracts, outstretched, one anteriorly one posteriorly; spermathecae small, round, empty. Anal body width 17.5 ± 2.7 (14.7 to 21.7) µm. Tail shape subcylindrical (Fig. 1c, f, h), 53.4 ± 2.4 (49.9 to 56.6) µm long, with 19–24 annules; tail terminus hemispherical, tail tip smooth. Males not found.

Fig. 1
figure 1

Tylenchorhynchus annulatus (Cassidy, 1930) Golden, 1971. a Entire female (rice population; arrows indicate vulva); b Female anterior end (stoma and oesophagus); c Female posterior end; d Entire female (corn population; arrows indicate vulva); e Female anterior end (stoma and oesophagus); f Female posterior end; g lateral lines; h Female posterior end; i Female anterior end. (Scale bar: a-f = 100 µm; g-i = 50 µm)

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The morphometrics of the Indonesian T. annulatus females isolated from corn are, in general, in agreement with the measurements of T. annulatus populations isolated in the USA, Nigeria, Malaysia, South Korea, China and Taiwan (Mizukubo and Toida 1991; Chen et al. 2006; Handoo et al. 2014; Pham et al. 2014; Mwamula et al. 2020; Abdulsalam et al. 2022). The Indonesian females are somewhat shorter in body length compared with the females from the T. annulatus populations described by Chen et al. (2006), Handoo et al. (2014), Pham et al. (2014), Mwamula et al. (2020) and Abdulsalam et al. (2022) but a shorter female body length was also reported for a T. annulatus population isolated from rice in Malaysia (515–741 µm) (Mizukubo and Toida 1991). An offset lip region is often considered an important diagnostic character of T. annulatus (Handoo 2000) but as mentioned by Mizukubo and Toida (1991) is often difficult to distinguish from a continuous lip region. The lip region of several populations isolated from various crops (corn, wheat and rice) on Nigeria was also described as slightly offset (Abdulsalam et al. 2022) while a continuous lip region was reported by Pham et al. (2014). Specimens of both T. annulatus populations were deposited in the collection of the Plant Pest Laboratory, Department of Plant Protection, Faculty of Agriculture, Universitas Gadjah Mada.

In addition to identification based on morphometrics and morphological characters, molecular identification was also carried out. DNA was extracted using the Geneaid™ DNA Extraction Kit (Tissue) protocol with modification on sample preparation. Amplification of the D2/D3 expansion region of 28S rDNA with forward primer D2A (5’-ACAAGTACCGTGAGGGAAAGTTG-3’) and reverse primer D3B (5’-TCGGAA GGAACCAGCTACTA-3’) (Nunn 1992) yielded a single fragment as targeted, approximately 780 bp. Sequences of the T. annulatus rice and corn populations were submitted to GenBank (accession numbers OP349594 and OP349600, respectively). The molecular characterization confirmed the identification of the Indonesian T. annulatus populations isolated from rice and corn based on morphometrics and morphological characterization. Nucleotide base comparison showed a very high percentage of similarity (96.5–99.7%) of the Indonesian T. annulatus populations with Chinese T. annulatus populations while the maximum likelihood tree of partial 28S rRNA placed the Indonesian T. annulatus populations closest to the T. annulatus populations from China and South Korea, separated from the T. annulatus populations from the USA (Fig. 2).

Fig. 2
figure 2

Partial 28S rRNA Maximum-Likelihood tree under General Time Reversible (GTR + G) model inferred from known and newly sequenced Tylenchorhynchus annulatus from Indonesia (in bold). Substitution model were selected based on the lowest Bayesian Information Criterion (BIC) scores. Models with the lowest BIC scores (Bayesian Information Criterion) are considered to describe the substitution pattern the best. ML tree were conducted in MEGA X

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Identification of Tylenchorhynchus populations is challenging because indications of the existence of species complexes within the genus. For instance, Mwamula et al. (2020) mentioned that there is a possibility that T. agri sequences submitted in GenBank could represent as T. annulatus. ITS-rRNA sequence studies by Mwamula et al. (2020) also revealed the existence of two types of T. annulatus (i.e. Type A and Type B). Based on the maximum likelihood tree of partial 28S rRNA, our samples clustered in a group of T. annulatus Type A as described by Mwamula et al. (2020). To our knowledge, this is the first report of T. annulatus in Indonesia.