Morphological and Molecular Characterization of Tylenchorhynchus clarus Allen, 1955 and T. zeae Sethi & Swarup, 1968 (Rhabditida: Telotylenchidae) from Iraq

The taxonomic framework proposed by Siddiqi (2000) was followed in this study, which considered Bitylenchus, Sauertylenchus Sher, 1974, and Tylenchorhynchus as valid genera.

For molecular analyses, single female specimens were picked out, examined in a drop of distilled water on a temporary slide under the light microscope, transferred to 5 μl of TE buffer (10 mM Tris-Cl, 0.5 mM EDTA; pH 9.0) on a clean slide, and then crushed using a cover slip. Each suspension was collected by adding 15 μl TE buffer. The DNA samples were stored at –20°C until they were used as PCR templates. Primers for LSU rDNA D2–D3 amplification were forward primer D2A (5'-ACAAGTACCGTGAGGGAAAGT-3') and reverse primer D3B (5'-TCGGAAGGAACCAGCTACTA-3') (Nunn, 1992). Primers for amplification of ITS rDNA were forward primer rDNA1 (5'-TTGATTACGTCCCTGCCCTTT-3') and reverse primer rDNA1.58S (5'-ACGAGCCGAGTGATCCACCG-3') (Subbotin et al., 2000). The 30 μl PCR mixture contained 10 μl of distilled water, 15 μl of Master Mix 2X (Ampliqon, Odenese, Denmark), 1 μl of each primer (10 pmol/μl), and 3 μl of DNA template. The thermal cycling program for amplification of both markers was as follows: denaturation at 95°C for 6 min, followed by 35 cycles of denaturation at 94°C for 30 s, annealing at 52.5°C (LSU D2–D3 primers) / 54.7°C (ITS rDNA primers) for 30 s, and extension at 72°C for 60 s. A final extension was performed at 72°C for 10 min. Amplification success was evaluated by electrophoresis on 1% agarose gel. The PCR products were purified using the QIAquick PCR purification kit (Qiagen, Hilden, Germany) following the manufacturer’s protocol and sequenced directly using the PCR primers with an ABI 3730XL sequencer (Bioneer Corporation, Daejeon, South Korea). The newly obtained sequences of the studied species were deposited into the GenBank database (accession numbers ON651683, ON651684, ON651685, ON651686 for LSU D2–D3, and ON667938, ON667939 for ITS rDNA).

The newly obtained sequences of the D2–D3 fragments of LSU rDNA and ITS rDNA and additional sequences of relevant species were selected after the nucleotide basic local alignment search tool (BLASTn, National Center for Biotechnology Information, Bethesda, Maryland, United States) search. The sequences were aligned by Clustal X version 2 using the default parameters (Larkin et al., 2007). The outgroup taxa were chosen according to a previous study (Abdolkhani and Azimi, 2021). The editing of both alignments was performed manually in the MEGA7 program (Kumar et al., 2016). The base substitution model was selected using MrModeltest2 (Nylander, 2004) based on the Akaike information criteria. A general time reversible model, including among-site rate heterogeneity and estimates of invariant sites (GTR + G + I), was used in both phylogenies. The Bayesian analysis was performed to infer the phylogenetic trees using MrBayes v3.1.2 (Ronquist and Huelsenbeck, 2003), running the chains for four million generations. After discarding burn-in samples and evaluating convergence, the remaining samples were retained for further analyses. The Markov chain Monte Carlo (MCMC) method within a Bayesian framework was used to determine equilibrium distribution and help estimate the posterior probabilities of the phylogenetic trees (Larget and Simon, 1999) using the 50% majority rule. Bayesian posterior probability (BPP) values >0.50 are given on appropriate clades. The output files of the phylogenetic program were visualized using Dendroscope v3.2.8 (Huson and Scornavacca, 2012) and were digitally drawn in CorelDRAW software version 17 (Corel Corporation, Ottawa, Canada).

Not found.

(Fig. 2 and Table 1)

Figure 2

The general morphology of the recovered population of the species closely resembles that of the type population (Sethi and Swarup, 1968). However, the length of the tail is shorter (25.2–38.2 vs 46 μm). This species has already been reported from Spain (Handoo et al., 2014), Iran (Alvani et al., 2017), and China (Xu et al., 2020). There is no significant difference between these populations and the population recovered from Misan province (southeast Iraq) as part of the current study. Compared to the morphometric data ranges given by Geraert (2011), the Iraqi population has a slightly shorter tail (25.2–38.2 vs 46 mm).

The presently studied species was recovered from the rhizosphere of pumpkin in Misan province, southeast Iraq (GPS coordinates: 32°07'56.81"N, 46°43'05.75"E). The present study is the first to report the discovery of T. zeae in Iraq, and additionally represents the first report of its association with pumpkin.