Morphological and Molecular Characterization of Quinisulcius curvus from China

Rhizosphere soil samples were collected from sea randa. Nematodes were extracted from soil and root samples of sea randa using the modified Cobb sieving and flotation-centrifugation method (Jenkins, 1964). Nematode suspension contained mixture of herbivores (Dorylaimids), fungivores (Aphelenchoides, Filenchus, Aphelenchus spp.) bacterivores (Rhabditis sp.) and populations of spiral and Quinisulcius sp. Since Quinisulcius was the most abundant species in the soil suspension, the females were collected individually from the mixture of soil nematodes and studied under light micrscope. For preliminary examinations, fresh Quinisulcius females were transferred to a drop of distilled water, heat relaxed and observed under a Zeiss microscope. For additional morphological and morphometric studies, nematodes were killed and fixed in hot formalin (4% formaldehyde) and processed to ethanol-glycerin dehydration according to Seinhorst (1959) as modified by De Grisse (1969) and mounted on permanent slides. Measurements were made on mounted specimens, light micrographs and illustrations were produced using a Zeiss microscope equipped with a Zeiss AxioCam MRm CCD camera.

DNA samples were prepared according to Li et al. (2008). Four sets of primers (synthesis by Invitrogen, Shanghai, China) were used in the PCR analyses to amplify sequences of the near full-length 18S region, D2-D3 expansion segments of 28S, and ITS of ribosomal RNA genes (rDNA). The 18S region was amplified with primers 988F/1912R and 1813F/2646R (Holterman et al., 2006). The 28S D2-D3 region was amplified with primers D2A/D3B (De Ley et al., 1999), and the ITS was amplified using primers TW81/AB28 (Tanha Maafi et al., 2003). PCR conditions were as described by Ye et al. (2007) and Li et al. (2008). PCR products were separated on 1% agarose gels and visualized by staining with ethidium bromide. PCR products with high quality were purified for cloning and sequencing by Invitrogen, Shanghai, China.

Sequenced DNA fragments from the present study (after discarding primer sequences and ambiguously aligned regions) and other stunt nematode sequences obtained from GenBank were used in the phylogenetic reconstruction. Outgroup taxa for each dataset were selected based on previously published studies (Handoo et al., 2014; Munawar et al., 2021; Nguyen et al., 2019). Multiple sequence alignments of the newly obtained and published sequences were made using the FFT-NS-2 algorithm of MAFFT V.7.450 (Katoh et al., 2019). Sequence alignments were visualized with BioEdit (Hall, 1999) and manually edited by Gblocks ver. 0.91b (Castresana, 2000) in the Castresana Laboratory server ( http://molevol.cmima.csic.es/castresana/Gblocks_server.html ) using options for a less stringent selection (minimum number of sequences for a conserved or a flanking position: 50% of the number of sequences + 1; maximum number of contiguous non-conserved positions: 8; minimum length of a block: 5; allowed gap positions: with half).

Phylogenetic analyses of the sequence datasets were conducted based on Bayesian inference (BI) using MRBAYES 3.2.7a (Ronquist and Huelsenbeck, 2003). The best-fit model of DNA evolution was calculated with the Akaike information (AIC) of JMODELTEST V.2.1.7 (Darriba et al., 2012). The best-fit model, the base frequency, the proportion of invariable sites, substitution rates and the gamma distribution shape parameters in the AIC were used for phylogenetic analyses. BI analyses were performed under a transitional model, with a rate of variation across sites (TIM3 + G) for the partial 28S region; a transversional model with a proportion of invariable sites and a rate of variation across sites (TVM + I + G) for ITS; and a transitional model with a proportion of invariable sites and a rate of variation across sites (TIM1ef + I + G) for 18S region. These BI analyses were run separately per dataset with four chains for 2 × 10⁶ generations. The Markov chains were sampled at intervals of 100 generations. Two runs were conducted for each analysis. After discarding burn-in samples of 30% and evaluating convergence, the remaining samples were retained for more in-depth analyses. The topologies were used to generate a 50% majority-rule consensus tree. Posterior probabilities (PP) are given on appropriate clades. Trees from all analyses were edited by FigTree software V.1.4.4 (Rambaut, 2014).

Quinisulcius curvus (Williams, 1960) Siddiqi, 1971

(=Tylenchorhynchus curvus Williams, 1960)

(Figures 1–4; Table 1)

Figure 1:

Figure 2:

Figure 3:

Figure 4:

Female

Body straight to slightly arcuate ventrally after heat fixation. Cuticle annulated and annuli becomes wider (1.0-1.5 µm) at mid-body. Lateral field with 5 incisures, non-areolated, extending about half of maximum body diameter, the middle incisures not extending past the phasmid; lip region hemispherical, bearing 4 to 7 fine annuli, slightly offset from body, 6.0 to 7.0  µm wide, 2.5 to 3.5  µm high; labial framework lightly sclerotized. Stylet knobs rounded, slightly posteriorly directed, 3.0 to 4.0  µm across. Dorsal gland orifice at 1.5 to 2.5  µm behind the stylet knobs. Median bulb oval, with conspicuous valve plates. Isthmus slender encircled with nerve ring. Excretory pore located at the middle of basal pharyngeal bulb, hemizonid 3 to 4 body annuli anterior to excretory pore. Basal pharyngeal bulb pyriform, abutting intestine. Cardia hemispherical. Reproductive system didelphic-amphidelphic, oocytes in single row. Vagina about half vulval body diam. deep, vulva depressed, apparently covered with a flap, spermatheca irregular inconspicuous, without sperm. Anus prominent. Tail cylindrical, with non-annulated bluntly rounded terminus, bearing 15 to 23 annuli on ventral side. Phasmids almost at middle of tail.

Not found.

Quinisulcius curvus was originally described from Mauritius in the rhizosphere of sugarcane (Williams, 1960). Since then, the species has been reported from Dominican Republic, Pacific Islands, Martinique, Thailand, India, Pakistan in the rhizosphere of sugarcane, grapes, maize, tuber and vegetable crops (Bohra and Baqri, 2005; Bridge, 1988; Cadet et al., 1994; Hussain et al., 2016; Mizukubo et al., 1993; Román, 1968; Toida et al., 1996). Though it has been reported from several countries but morphometrical and morphological data was only provided in few reports (Table 1). Morphometrically, it is observed that the Hainan population of Q. curvus has a shorter stylet than the type population (11.5-13.5 µm vs 17.0 µm) and other reported descriptions (Table 1), but close to a Thailand population (11.5-13.5 µm vs 13.5-15 µm) (Mizukubo et al., 1993), suggesting a high intraspecific variability on this character. However, the rest of morphometrics and morphological characters e.g. body habitus, lip and tail morphology, and lateral field characters posterior to the phasmid correspond well with the original description. Vulva and spermatheca morphology was not described in the original or subsequent descriptions. The vulva of the Hainan population has fine lips and apparently covered with a vulval flap. The spermatheca is non-functional, weakly developed and irregularly shaped. No sperm was observed in the spermatheca. Male was not described in the original description or in the subsequent descriptions (Li et al., 1986; Mizukubo et al., 1993; Williams, 1960), same as in the Hainan population. Out of 17 nominal species of Quinisulcius, males were not reported for 10 species (Geraert, 2011). It can be speculated that Q. curvus is a parthenogenetic species as evidenced by the empty spermatheca.

In broader sense, the species of Quinisulcius are medium to large worms; their body length ranges from 460 to 911 µm. The shortest species is Q. quaidi (Zarina and Maqbool, 1992) (744-911 µm) and the longest is Q. capitatus (830-960 µm). The general body habitus is arcuate to C-shape, however, Q. seshadrii (Giribabu and Saha, 2002) has spiral body habitus. The stylets of Quinisulcius species are moderately strong, ranging from 11 to 23 µm. The shortest stylet was reported for Q. quaidi (11-13 µm) and the longest for Q. dalatensis (Nguyen and Nguyen, 1998) (21-23 µm). The excretory pore position is quite variable; however, it is always located in the region of the pharyngeal bulb or anterior to it. The tail is variable, with a length ranging from 30 to 58 µm, the longest of which was reported for Q. brevistyletus (Kulinich, 1985) (51-58 µm). The general morphology of the tail is elongate conoid and ventrally curved with rounded to pointed terminus (Geraert, 2011). Our species in question, Q. curvus is morphologically close to Q. acutus and Q. capitatus. It can be differentiated from both species by smaller body size, smaller stylet and shape of tail region. Q. curvus differs from Q. acutus by lip morphology (hemispherical continuous without constriction vs rounded set off by constriction), stylet knobs (rounded vs massive cupped-shaped), cardia shape (hemispherical vs conoid-rounded), shorter body (581 (503-644) µm vs 600-750 µm), and shorter stylet (12.6 (11.5-13.5) µm vs 15-17 µm). From Q. capitatus, it can be distinguished by the shape of lip region (hemispherical vs rounded), shorter body (581 (503-644) µm vs 744.0-911.0 µm), and shorter stylet (12.6 (11.5-13.5) µm vs 15.5-20.4 µm).

This population was collected in the rhizosphere of sea randa (Guettarda speciosa L.) from Ganquan Island, Sansha City, Hainan Province, China on September 23, 2019 (6.509068N, 111.596901E).

The sequences of nearly full-length 18S (1649 bp, NW628178-NW628179), ITS region of rDNA (806 bp, NW628172-NW628173) and 28S D2-D3 region (742 bp, NW628174-NW628175) of Q. curvus were obtained in the present study. Phylogenetic relationships among the subfamily Merliniinae and subfamily Telotylenchinae nematodes were determined separately for each dataset using BI (Figs. 2-4). These subfamilies were selected because of the genus Quinisulcius is morphologically close to both of them.

So far, Q. capitatus is the only species of the genus that has been molecularly characterized. The sequence identities of partial 18S, ITS and 28S rDNA of Q. curvus with Q. capitatus are 95% (34 bp difference, 0 indel), 82% (99 bp difference, 44 indels) and 89% (75 bp difference, 10 indels), respectively, confirming the species separation.

The phylogenetic analyses of Q. curvus are presented in Figures 2-4. The trees inferred from 18S, and 28S analyses separated into two distinct clades (PP = 1.00, 1.00, respectively) which comprises species of subfamilies Telotylenchinae and Merliniinae; however, in ITS tree Telotylenchinae appears separate in three different groups (Fig. 4). In all the trees, both Quinisulcius species (Q. capitatus and Q. curvus) are clustered together and grouped with members of Telotylenchinae, except for 18S tree (Fig. 2), in which both species appeared on separate branches among species of Tylenchorhynchus. However, in 28S and ITS trees (Figs. 3 and 4), Q. curvus and Q. capitatus grouped together as a subclade within species of Tylenchorhynchus. We also anticipate that with the inclusion of more sequences from Quinisulcius and species from other genera within these subfamilies will certainly clarify the phylogenetic positioning of Q. curvus and Q. capitatus, as well as the monophyletic or paraphyletic status of the genus Quinisulcius and the subfamilies Merliniinae and Telotylenchinae.

Our results suggest that Merliniinae and Telotylenchinae appears clearly separated with 18S and 28S ribosomal markers (Figs. 2 and 3), confirming morphological separations, and appearing as monophyletic separated groups. Nevertheless, in ITS Telotylenchinae appears as a polyphyletic group (Fig. 3). Consequently, additional species characterization under integrative taxonomic approaches are needed to confirm this hypothesis by studying several species of other genera within these subfamilies.

The host associations or preference of Q. curvus has not been well documented. It was initially reported from the sugarcane rhizosphere in Mauritius (Williams, 1960), subsequently it has been detected in sugarcane growing areas of Martinique and Australia (Nobbs, 2013; Román, 1968). From these reports, it can be speculated that Q. curvus has a host preference for sugarcane. In addition to that, Q. curvus has also been reported from other countries in the rhizosphere of different horticultural crops, although no considerable plant damage was reported (Table 1). Quinisulcius curvus was also reported in Henan Province of China from the rhizosphere of Brassica caulorapa, but the diagnosis of this population was based on a single specimen (Li et al., 1986, Table 1). In our opinion, a morphological identification based on single nematode is doubtful. Therefore, the status of the Henan population of Q. curvus needs further sampling and confirmation.

It has been noted that Q. curvus has always been reported in association with principle agricultural or horticultural crops. In this study, we found Q. curvus in the rhizosphere of shrub sea randa i.e. G. speciosa, which is a new host record for this species. The discovery of Q. curvus from China highlights the need to update the list of distribution of these nematodes. Moreover, comprehensive surveys will likely uncover other Quinisulcius species from China.