Introduction

Tomato (Lycopersicum esculentum L.) is one of the most popular vegetable crops in South Africa, and Limpopo is the major production area with approximately 3590 ha (Bozo et al. 2019). However, tomatoes are threatened by various pests and diseases. The genus Bitylenchus belongs to the family Dolichodoridae Chitwood in Chitwood and Chitwood, 1950. The genus Bitylenchus was controversial among several scientists (Andrássy 2007; Handoo et al. 2014). This genus has been synonymized with Tylenchorhynchus (Geraert 2011). However, using molecular analysis, Handoo et al. (2014) and Hoseinvand et al. (2020) considered it a valid taxon. Siddiqi (2000) evaluated 29 nominal species under the genus Bitylenchus. The genus Bitylenchus was distinguished from a close genus, Tylenchorhynchus, in having areolated outer bands of lateral fields, a large post-anal intestinal sac containing intestinal granules and fasciculi, a relatively more thickened cuticle at the female tail tip and gubernaculum lacking a crest (Handoo et al. 2014). However, their ecological behavior and crop damage are not well understood. During a survey on the nematodes of South Africa in Limpopo Province, B. ventrosignatus (Tobar Jiménez, 1969) Siddiqi, 1986 was recovered from a tomato in Dalmada. As tomato is among the most economical crop in South Africa, the effect of Bitylenchus on tomato production becomes essential. Besides, diagnosis of this nematode is crucial. Previously, B. ventrosignatus has been reported in association with Banana in South Africa (Jones 1979); however, no morphological and molecular support was provided. Therefore, the study’s aims were (1) to study the morphology of B. ventrosignatus, and (2) to study the phylogenetic position of South African B. ventrosignatus with the related species.

Materials and methods

Nematode extraction and processing

The soil sample was collected from the rhizosphere of a tomato in Dalmada, Limpopo Province, South Africa (GPS coordinates: S: 23°53’44.411”; E: 29°32’55.192”) (Fig. 1a-c). Nematode extraction was done using the modified tray method (Shokoohi et al. 2023). Extracted nematodes were fixed with a hot 4% formaldehyde solution (except those specimens used for molecular analyses), transferred to anhydrous glycerin utilizing the method of De Grisse (1969), and mounted on permanent glass slides. The classification provided by Handoo et al. (2014) was used for the taxonomic study of Bitylenchus.

Fig. 1
figure 1

Sampling location in Dalmada, Limpopo Province, South Africa. a South Africa map; b Dalmada region of Limpopo Province (arrow pointing to sampling site); c tomato field where sampled

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Light microscopy (LM)

Measurements of specimens mounted on permanent slides were taken, and De Man’s (1881) indices were calculated. Drawing was done using a camera attached to a Zeiss microscope (Axio Lab) at the Aquaculture Research Unit, University of Limpopo. Micrographs were taken under a Nikon Eclipse 80i light microscope with differential interference contrast optics (DIC) (Nikon, Tokyo, Japan). Micrographs were edited using Adobe® Photoshop® CS.

DNA extraction, PCR, and phylogenetic analysis

DNA extraction was done using the Chelex method (Shokoohi et al. 2023). Five specimens of each species were hand-picked with a fine-tip needle and transferred to a 1.5 ml Eppendorf tube containing 20 µl double distilled water. The nematodes in the tube were crushed with the tip of a fine needle and vortexed. Thirty microliters of 5% Chelex® 50 and 2 µL of proteinase K were mixed into the microcentrifuge tube containing the crushed nematodes. The microcentrifuge tube with the nematode lysate was set at 56 °C for two hours and then incubated at 95 °C for 10 min to deactivate the proteinase K and finally spin for 2 min at 16,000 rpm (Shokoohi and Abolafia 2023). The supernatant was extracted from the tube and stored at − 20 °C. Following this step, the forward and reverse primers, SSU F04 (5’-GCTTGTCTCAAAGATTAAGCC–3’) and SSU R26 (5’-CATTCTTGGCAAATGCTTTCG–3’) (Blaxter et al. 1998) for 18S rDNA and D2A (5’-ACAAGTACCGTGAGGGAAAGTTG–3’), D3B (5’-TCGGAAGGAACCAGCTACTA–3’) (De Ley et al. 1999) for 28S rDNA, were used in the PCR reactions for amplification of the 18S rDNA, and 28S rDNA regions. PCR was conducted with eight µl of the DNA template, 12.5 µl of 2X PCR Master Mix Red (New England Biolabs; NEB), one µl of each primer (10 pmol µl-1), and ddH2O for a final volume of 30 µl. The amplification was processed using an Eppendorf master cycler gradient (Eppendorf, Hamburg, Germany), with the following program: initial denaturation for 3 min at 94 °C, 37 cycles of denaturation for 45 s at 94 °C; 54 and 56 °C annealing temperatures for 18S rDNA and 28S rDNA, respectively; extension for 45 s to 1 min at 72 °C, and finally an extension step of 6 min at 72 °C followed by a temperature on hold at 4 °C. After DNA amplification, four µl of product from each tube was loaded on a 1% agarose gel in TBE buffer (40 mM Tris, 40 mM boric acid, and 1 mM EDTA) for evaluation of the DNA bands. The bands were stained with ethidium bromide and visualized and photographed on a UV transilluminator. The amplicons of each gene were stored at − 20 °C. Finally, Inqaba Biotech (South Africa) purified the PCR products for sequencing. Available sequences for other Bitylenchus spp. were obtained from NCBI GenBank for comparison. Also, as outgroups, Coslenchus costatus (de Man, 1921) Siddiqi, 1978 (KX156285; DQ328719) based on Shokoohi (2021), were used as the outgroup for the 18S and 28S rDNA analyses, respectively. The ribosomal DNA sequences were analyzed and edited with BioEdit (Hall 1999) and aligned using CLUSTAL W (Thompson et al. 1994). Phylogenetic trees were generated using the Bayesian inference method implemented in the program Mr. Bayes 3.1.2 (Ronquist and Huelsenbeck 2003). The GTR + I + G model was selected using jModeltest 2.1.10 (Guindon and Gascuel 2003; Darriba et al. 2012). Then, the chosen model was initiated with a random starting tree and ran with the Markov chain Monte Carlo (MCMC) for 106 generations. The trees were visualized using FigTree v1.4.4 (Rambaut 2018).

Results

Bitylenchus ventrosignatus (Tobar Jiménez, 1969) Siddiqi, 1986

(Figs. 2 and 3; Table 1)

Table 1 Measurements of B. ventrosignatus from South Africa
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Fig. 2
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Bitylenchus ventrosignatus (Tobar Jiménez, 1969) Siddiqi, 1986. a Neck; b anterior end (stylet and dgo); c entire male; d female posterior end; e entire female; f female reproductive system; g male posterior end; h irregular vaginal undulation

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Fig. 3
figure 3

Bitylenchus ventrosignatus (Tobar Jiménez, 1969) Siddiqi, 1986 (LM). a Entire male; b entire female (arrow pointing to vulva); c-e anterior end; f neck; g male posterior end; h vagina region (arrow pointing to undulation); i female posterior end (arrow pointing to anus)

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Female (n = 7; Table 1): Body almost open C-shaped or straight after heat relaxation (Figs. 2e, 3b), no longitudinal striae or ridges outside lateral fields. Body annuli distinct, 0.9–1.1 μm wide around mid-body. Lateral fields originating at the level of the conus of the stylet and extending up to hyaline region of tail terminus, with four incisures, 21–38% of the corresponding body diameter. Lip region high, spherical, offset to body contour (Figs. 2a, b, 3c-e), 3.8 ± 0.4 (3–4) µm height, 7.7 ± 0.8 (6–9) µm diameter; with four to five annuli. Conus 47–50% of the stylet length, shaft 39–41% of the stylet length, and knobs 12–15% of the stylet length. Stylet moderately strong (Figs. 2b, 3c-e), conus slightly longer than shaft; knobs laterally to posteriorly directed. Dorsal gland orifice about 1.5-3.0 μm long behind stylet base. Median pharyngeal bulb oval; basal bulb pyriform. Cardia well-developed. Hemizonid usually just two to three annuli anterior to excretory pore, 1.0–1.5 annuli wide. Vulva a transverse slit slightly posterior to the middle of the body, vagina with 8.7 ± 0.8 (6–11) µm length. Epiptygma absent. Cuticle posterior to vulva with undulation (Figs. 2h, 3h). Reproductive system amphidelphic, didelphic; anterior (161.7 ± 15.7 (146–183) µm) and posterior (165.5 ± 32.1 (127–205) µm), ovaries well developed (Fig. 2f). Spermatheca rounded, filled with rounded spermatozoa. Tail subcylindrical (Figs. 2d, 3i), tail terminus rounded and smooth. Phasmids located slightly anterior to middle of the tail, 34–44% of tail length. Post anal intestinal sac present.

Male (n = 5; Figs. 2c, 3a; Table 1): Body J-shaped or S-shaped after relaxation. The morphology similar to the females, except for the reproductive system. Testis one, outstretched anteriorly. Spicules tylenchoid, paired and symmetrical, 8–9 times longer than wide: slightly elongate and ventrally curved, rounded manubrium, very short and straight calamus, and ventrad curved lamina with an acute tip, bursa large and conspicuous, extending to tail tip, 61–71 μm long. Gubernaculum well developed, curved, about 27–40% of the spicule length. Tail terminus rounded (Figs. 2g, 3g).

Phylogenetic analysis (Figs. 4, 5): The original partial 18S rDNA and 28S rDNA (D2-D3 expansion) sequences of B. ventrosignatus were deposited in GenBank under the accession numbers OR035484 (18S rDNA) and OR035418 (28S rDNA). The Bayesian inference tree of 18S rDNA of Bitylenchus species (Fig. 4) placed the South African B. ventrosignatus close to Botswana population of B. ventrosignatus (acc. nr: MW255611) with 1.00 posterior probability. Besides, the Bayesian tree of 28S rDNA (Fig. 5), placed South African B. ventrosignatus close to the Botswana (acc. nr: MW255613) population of B. ventrosignatus with 1.00 posterior probability. Additionally, all populations of B. ventrosignatus form a group with a 1.00 posterior probability.

Fig. 4
figure 4

The Bayesian tree inferred from known and newly sequenced Bitylenchus ventrosignatus from South Africa based on the 18S rDNA region

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Fig. 5
figure 5

The Bayesian tree inferred from known and newly sequenced Bitylenchus ventrosignatus from South Africa based on the 28S rDNA region

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Discussion

Bitylenchus is an ectoparasite nematode that has been reported in association with bananas in South Africa (Jones 1979). However, no evidence of morphology was available from South Africa. Therefore, the present study is the first report on the morphological and molecular of 18S and 28S rDNA. The morphological characters of the studied population of B. ventrosignatus fit with the previous material of the same species studied (Fortuner and Luc 1987; Geraert 2011). However, Fortuner and Luc (1987) reported B. ventrosignatus lacks a postanal intestinal sac, the character observed in the South African specimens. The Botswanan specimens also bear this feature (Shokoohi 2021). Compared with the material examined by Handoo et al. (2014), samples from South Africa have smaller female body length (585–626 vs. 610–722 μm), fewer tail annuli (30–35 vs. 32–42) and smaller gubernaculum (6–10 vs. 10–12 μm). Compared with the material examined by Geraert et al. (1975), they differ in the female stylet length (10–14 vs. 12.5–15 μm), female neck length (111–126 vs. 98–121 μm), and male body length (598–621 vs. 560–580 μm). In addition, the South African B. ventrosignatus showed irregular undulation in the vulva region, a character that has been described in the original description for B. ventrosignatus (Tobar Jiménez 1969; Geraert et al. 1975; Geraert 2011). Compared with the Botswana population of B. ventrosignatus (Shokoohi 2021), they differ in body length (585–626 μm in females and 598–621 μm in males vs. 496–583 μm in females and 470–521 μm in males), and bursa length (61–71 vs. 59–76 μm).

The 18S and 28S rDNA grouped the South African B. ventrosignatusi with another population of the same species, consequently confirming the species. The same result was obtained by Handoo et al. (2014) and Shokoohi (2021). The result also indicated B. ventrosignatus as a monophyletic group. However, the morphological variation may be detected due to the samples’ geographical position.

On the other hand, the phylogenetic analysis demonstrated that the genus Bitylenchus is not a monophyletic group. This is in agreement with Handoo et al. (2014). Besides, the results obtained by Hosseinvand et al. (2020) and Shokoohi (2021) indicated that Bitylenchus species divide into two groups. However, more sequences should be included in the phylogenetic study, albeit the species identification of the genus Bitylenchus is problematic.

From an economic point of view, this species is not common in South African soil. However, because of the crop’s economic importance, its presence in the tomato field requires an ecological and parasitological study of B. ventrosignatus on tomato production.

Therefore, using other DNA markers along with the SEM will reveal the actual position of the species belonging to Bitylenchus. Two permanent microscope slides containing seven females and five males were deposited in the Nematology collection of the Aquaculture Research Unit of the University of Limpopo, South Africa. Relative to published literature, this is the first record of B. ventrosignatus from tomato in South Africa.