First observation on morphological and molecular characters of Bitylenchus ventrosignatus (Tobar Jiménez, 1969) Siddiqi, 1986 isolated from tomato in Dalmada, South Africa

During a survey on the biodiversity of plant-parasitic nematodes in Limpopo Province, South Africa, Bitylenchus ventrosignatus was discovered around the rhizosphere of a tomato in Dalmada. The nematodes were extracted using the tray method and then fixed according to available protocols. The morphological characters fit well with the description of B. ventrosignatus. In addition, molecular analysis using 18S and 28S rDNA indicated 99% similarity (MW255611; MW255613) with the Botswana population of B. ventrosignatus. The phylogenetic analysis of 18S and 28S rDNA placed the examined population with other populations of B. ventrosignatus in a group with a posterior probability support value of 1.00. According to published literature, this is the first morphological and molecular characters of 18S and 28S rDNA of B. ventrosignatus from South Africa.


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

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. 1ac).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.

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'-ACAAGTACCGT-GAGGGAAAGTTG-3'), D3B (5'-TCGGAAGGAAC-CAGCTACTA-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 ddH 2 O 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 10 6 generations.The trees were visualized using FigTree v1.4.4 (Rambaut 2018).
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.
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Fig. 1
Fig. 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

Table 1
; Table 1) 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 Measurements of B. ventrosignatus from South Africa