Morphological and molecular characterisation of Helicotylenchus zengchengensis n. sp. (Nematoda: Hoplolaimidae) from China, with a review on Helicotylenchus species in China

Abstract

Helicotylenchus comprises a significant group of plant-parasitic nematodes that primarily feed on plant roots. This study focuses on the description of a newly discovered species, Helicotylenchus zenchengensis n. sp., from China. The distinguishing characteristic of this species, as observed under a microscope, is the absence of head annulation. The stylet, measuring between 23.8 and 29.3 μm, exhibits anteriorly flattened or indented stylet knobs. The elongated tail has a c value ranging from 15.8 to 22.6 and a c’ value between 1.9 and 2.4. It bears 15 to 18 tail annuli, presenting a conical shape with projections. The phasmids are located at the 4th to 10th body annulations posterior to the anus, while no males were observed. Three phylogenetic trees were constructed to determine the evolutionary relationships using the 18S rRNA, ITS, and 28S D2-D3 expansion segments of the newly discovered species and related Helicotylenchus sequences obtained from the database. Based on the ITS sequences, a phylogenetic tree revealed that H. zenchengensis n. sp. clustered with H. cuspicaudatus. However, it was distinguished from H. cuspicaudatus when examining the 18S and 28S rRNA sequences and morphological characteristics. Additionally, comprehensive and diagnostic tables for all known species of Helicotylenchus in China are provided for reference.

Background

The genus Helicotylenchus Steiner, 1945 belongs to the family Hoplolaimidae Filipjev, 1934, the infraorder Tylenchomorpha De Ley and Blaxter, 2002, and the suborder Tylenchina Thorne, 1949 (Decraemer and Hunt 2013). Nematodes in this genus are commonly known as spiral nematodes due to their spiral-shaped body in a relaxed and dead state, making them easily identifiable under a stereomicroscope (Crow 2013). There are over 230 valid species of Helicotylenchus, widely distributed worldwide and associated with many important crops (Uzma et al. 2015; Zameleh et al. 2020). Most species are ectoparasites, while a few are semi-endoparasites or occasional endoparasites. Some species, such as H. pseudorobustus (Steiner, 1914) Golden, 1956, H. dihystera (Cobb, 1896) Sher, 1961, and H. multicinctus (Cobb, 1893) Golden, 1956, can cause significant damage to crops (Subbotin et al. 2015; Rybarczyk-Mydłowska et al. 2019).

The morphological diagnostic characteristics of the genus Helicotylenchus include head shape, head annuli, stylet length, tail shape, and phasmid position relative to the anus. However, many species share similar diagnostic characteristics. Uzma et al. (2015) conducted a comprehensive study on the genus Helicotylenchus, providing diagnostic tables and line drawings for 224 species. Molecular identification and phylogenetic analyses based on conserved rRNA gene sequences have become powerful tools for species identification (Subbotin et al. 2011; Shokoohi et al. 2018; Rybarczyk-Mydłowska et al. 2019).

The first comprehensive work on the genus Helicotylenchus in China was conducted by Liu (2005), who provided important information on the morphology, localities, hosts, and references of 47 species in his book. Based on Liu’s work, subsequent publications have revealed that 55 species have been recorded in China, with only H. membranatus Xie & Feng 1993 being the new species described from this country. In 2021, a new species of Helicotylenchus was discovered in soil samples collected from the bamboo rhizosphere in the Zengcheng District, Guangzhou, Guangdong Province, China. This study aimed to describe the new species morphologically and molecularly, analyze its phylogenetic relationship with other Helicotylenchus species based on rRNA gene sequences, and provide comprehensive and diagnostic tables for all Helicotylenchus species in China.

Results

Helicotylenchus zengchengensis n. sp.

Measurements of holotype female and 12 paratype females are listed in Table 1 and Figs. 1, 2, 3, 4.

Table 1 Morphometric data of the females of Helicotylenchus zengchengensis n. sp

Fig. 1

Line drawings of female Helicotylenchus zengchengensis n. sp. a and b. Entire body; c and d Head region; e Pharynx; f Vulva region; g–j Tails

Fig. 2

Photomicroscopy of female Helicotylenchus zengchengensis n. sp. a and b Female entire body; c and d Anterior part; e Oesophageal part; f Genital part; g and h Incisures at middle body; i Vulva region

Fig. 3

Photomicroscopy of tails of female Helicotylenchus zengchengensis n. sp

Fig. 4

SEM photos of female Helicotylenchus zengchengensis n. sp. a and c Head; b Incisures in middle body; d–f Tails

Description:

Female. Body usually curved ventrally, forming spirals when killed by gentle heat, occasionally open 6-shaped. Body annuli 1.3–1.6 μm wide at mid-body. The lateral field bearing four incisures with outer margins slightly crenate, with no areolation except on the cuticle in the oesophageal part and occasionally seen in the tail end; the two middle incisures were further apart than the two outer incisures and formed a U- or Y-shaped pattern in the tail end, about 1/5th–1/4th mid-body width in the middle body. Labial disc fused on ventral and dorsal sides without lateral lips, and sub-oval oral disc with oblong opening in en face view. The cephalic region was elevated and flattened anteriorly, continuous with the body. Head annulations were hardly seen under a light microscope (LM), but 1–2 annulations close to the first annulation of the body were seen under a scanning electron microscope (SEM). The cephalic framework is heavily sclerotised. Stylet robust, 23.8–29.3 μm long; stylet metenchium 12.2–14 μm long, approximately half the length of stylet; stylet knobs rounded, anteriorly dented, or slightly flattened. Orifice of the dorsal oesophageal gland 9.5–13.4 μm posterior to stylet base. Median bulb oval, 8.3–13.3 μm × 7–9.7 μm with a prominent valvular apparatus. The oesophageal gland overlapped and wrapped over the anterior end of the intestine and the ventral side longest. Hemizonid located at 0–2 annuli anterior to the excretory pore. Excretory pore located above the level of the anterior part of oesophageal gland, 85.9–108 µm apart from anterior end. Genital tract amphidelphic, outstretched, developing oocytes arranged in a single row, spermatheca subrounded. Vulva transverse, not protuberant, lateral vulval flaps absent, at 322.3–428.3 µm apart from anterior end. Tail 27.8–33.5 μm long, with 15–18 annuli, asymmetrical conical with variable ventral projections. Phasmids were rounded, located at 4–10th body striations or 5–14 µm posterior to the anus.

Male. Not found.

Type locality and habitat

The new species was recovered from the soil associated with bamboo in Zengcheng District, Guangzhou, Guangdong Province, China. (N23.449877°N, 113.847389°E).

Etymology

The species name “zengchengensis” refers to the geological name where the new species was found.

Type material

Holotype and 11 paratype females were deposited at the Laboratory of Plant Nematology, South China Agricultural University, Guangzhou, Guangdong Province, and two paratype females were deposited in National Parasitic Resources Center in Shanghai, China.

Diagnose and relationships

Helicotylenchus zengchengensis n. sp. is characterized by the absence of head annulations under light microscopy (LM) and the presence of 1–2 annulations close to the first body annulation visible under scanning electron microscopy (SEM). The stylet length ranges from 23.8–29.3 μm with anteriorly flattened or indented stylet knobs. The lateral field bears four incisures, with the inner two incisures positioned farther apart from the outer ones, and areolations present in the oesophageal region. The tail is long and asymmetrical conical with projections, consisting of 15–18 annuli. Phasmids are located at the 4–10th body annulations or at a distance of 5–14 µm posterior to the anus. No males were found.

Helicotylenchus zengchengensis n. sp. can be readily distinguished from most species in the genus Helicotylenchus based on three diagnostic characters: the absence of head annulations under LM, the posterior position of the phasmids relative to the anus, and a long tail. It shares similarities with H. belli (Sher, 1996), H. ferus (Eroshenko & Nguen Vu Thanh, 1981), H. marethae (Marais, Queneherve, Tiedt & Meyer, 2013), and H. martini (Sher, 1996) based on the former three diagnostic characters. However, it differs from H. belli mainly in tail shape (asymmetrical conical with a ventral projection vs. hemispherical without projection), shorter body length (496.5–688.9 μm vs. 710–980 μm), and lower c value (15.8–22.6 vs. 27–48). It differs from H. ferus primarily in tail shape (asymmetrical conical with a ventral projection vs. hemispherical without projection), the number of body annuli from anus to phasmids (4–10 vs. 3–5), longer body length (496.5–688.9 μm vs. 470–490 μm), higher a value (27.8–32.9 vs. 21–23), lower c value (15.8–22.6 vs. 43–45), and longer stylet length (23.8–29.3 μm vs. 22–23 μm). It differs from H. marethae mainly in tail shape (asymmetrical conical and ventrally curved with a ventral projection vs. dorsally curved), lower c value (15.8–22.6 vs. 25.3–39.7), and higher “c” value (1.9–2.4 vs. 1.1–1.6). It differs from H. martini primarily in tail shape (asymmetrical conical with a ventral projection vs. smoothly rounded terminus) and a higher number of tail annuli (15–18 vs. 10).

Molecular characterization and phylogenetic analysis

In the study, molecular analysis was conducted on Helicotylenchus zengchengensis n. sp. Using conserved primers for the amplification of 18S partial rRNA segments, ITS, and the 28S D2-D3 expansion segments. The obtained sequences were deposited in GenBank with accession numbers OP874598 for 18S, OP828612–13 for ITS, OP828614, OQ912904, and OQ911481–82 for 28S D2-D3 expansion.

The assays using the Basic Local Alignment Search Tool (BLAST) revealed that the 18S rRNA gene sequence of H. zengchengensis n. sp. had the highest similarity (97.31%) to an unidentified Helicotylenchus species (Accession: MK292128) from rhizosphere soil of bamboo in America (Carta and Li 2019), followed by H. dihystera (Accession: AJ966486) with 96.57% identity, extracted from coastal soil in the United Kingdom (Meldal et al. 2007). A phylogenetic analysis of the 18S rRNA sequences (Fig. 5) showed that H. zengchengensis n. sp. is a unique species and formed a sister group with the unidentified species (Accession: MK292128), H. dihystera, H. digitiformis Ivanova, 1967, H. pseudorobustus, H. certus Eroshenko & Nguen Vu Thanh, 1981, H. multicinctus, H. crenacuada Sher, 1966, H. paraplatyurus Siddiqi, 1972, and H. indicus.

Fig. 5

Phylogenetic relationships of Helicotylenchus zengchengensis n. sp. with the species of the genus Helicotylenchus. The phylogenetic tree was constructed by Bayesian analysis using 18S rRNA partial sequences, with the optimal model GTR + I + G under AIC standard. Posterior probability values exceeding 50% are given on appropriate clades

Regarding the two ITS sequences from two individuals, there were 27 base pair variations. BLAST search results showed that these sequences had 96.91% and 97% identity with a sequence from H. cuspicaudatus (Accession: MG696764) extracted from the rhizosphere soil of bamboo in China. Phylogenetic analysis of the ITS sequences (Fig. 6) confirmed that the two sequences of H. zengchengensis n. sp. clustered together and formed a well-supported clade with H. cuspicaudatus (PP = 100%) and separated from other species. The four 28S rRNA gene sequences obtained from four individuals of H. zengchengensis n. sp. were all 791 bp long, exhibiting only three base pairs of variation. The BLAST results indicated that these sequences were most closely related to a sequence from an unidentified Helicotylenchus species (Accession: ON117611) with a 99.9% identity, and the species was extracted from rhizosphere soil of bamboo in Fujian Province, China. The Bayesian phylogenetic analysis of the 28S rRNA sequences (Fig. 7) revealed that the four sequences of H. zengchengensis n. sp. and the sequence of Accession ON117611 formed a distinct clade (PP = 100%) which is closely related to a sister group consisting of H. asiaticus Mwamular, Ma, Kim, Kim, Han & Lee, 2020 and the unidentified species (Accession: KY484830 and KY484825), but are less related with the three sequences from H. cuspicaudatus (Accession: MG696758–60).

Fig. 6

Phylogenetic relationships of Helicotylenchus zengchengensis n. sp. with the species of the genus Helicotylenchus. The phylogenetic tree was constructed by Bayesian analysis using ITS-rRNA gene sequences, with the optimal model GTR + I + G under AIC standards. Posterior probability values exceeding 50% are given on appropriate clades

Fig. 7

Phylogenetic relationships of Helicotylenchus zengchengensis n. sp. with the species of the genus Helicotylenchus. The phylogenetic tree was constructed by Bayesian analysis using 28S rRNA D2-D3 gene sequences, with the optimal model GTR + I + G under AIC standards. Posterior probability values exceeding 50% are given on appropriate clades

Although the new species clustered with H. cuspicaudatus based on ITS-rRNA sequences and showed the highest identity with H. cuspicaudatus, it did not group with H. cuspicaudatus in the same clade based on the 28S rRNA gene sequences. In addition, it differed from H. cuspicaudatus in terms of morphological characteristics. These differences include a distinct lip annulus (smooth under LM vs. 4), a different location of phasmids (4–12 annuli posterior to anus vs.5 annuli anterior to anus), a lower c value (15.8–22.6 vs. 36–33), and a higher c′ value (1.9–2.4 vs. 1.1–1.8).

A diagnostic table of Helicotylenchus species in China

In China, 55 species have been described and redescribed. A table containing information on hosts and localities of 56 Helicotylenchus species in China (Table 2) is presented to facilitate the analysis of their geological and host distributions for further study. Among these species, H. dihystera stands out as the most frequently redescribed species with the broadest distribution and host range in China, followed by H. digonicus Perry in Perry, Darling and Thorne, 1959 and H. pseudorobustus. In addition, H. califormicus Sher, 1966, H. multicinctus, H. erythirinae (Zimmermann, 1904) Golden, 1956, H. exallus Sher, 1966, H. minzi Sher, 1966, H. abunaamai Siddiqi, 1972, and H. crenacauda Sher, 1966 have widespread distributions, spanning five provinces (autonomous regions/cities) in China. For ease of species identification, a diagnostic table including information on the 56 species is provided in Table 3, and the diagnostic characters used for identification are consistent with those given by Uzma et al. (2015).

Table 2 Hosts and localities of Helicotylenchus species in China

Table 3 Diagnostic data on species of the genus Helicotylenchus females in China (data all from original description)

Discussion

The genus Helicotylenchus comprises a large family with 230 valid species. Species identification based on morphological studies is challenging due to shared diagnostic characteristics and significant intraspecific variation (Subbotin et al. 2011; Uzma et al. 2015). Molecular identification has proven to be a powerful tool, but most species have no sequencing data. Subbotin et al. (2011) conducted an extensive phylogenetic analysis of 54 isolates from 10 species, utilizing 89 sequences of the 28S rRNA D2-D3 expansion. They further expanded their research with another comprehensive study (Subbotin et al. 2015), focusing on six species within Helicotylenchus, and constructed phylogenetic trees using 154 sequences of 28S D2-D3 and 37 sequences of ITS. Their findings demonstrated that species could be distinguished from one another, although some species formed paraphyletic groups, and certain sequences submitted to GenBank were potentially misidentified. Subbotin et al.’s conclusions were further supported by Shokoohi et al. (2018) and Rybarczyk-Mydłowska et al. (2019). These researchers conducted comprehensive investigations involving numerous isolates from three different species. They combined the 28S D2-D3 expansion segments with morphological characteristics in their studies. It is noteworthy that, although over 20 species within Helicotylenchus were sequenced, the majority of DNA sequences available in GenBank pertain to only a few species, such as H. dihystera, H. pseudorobustus, and H. microlobus Perry in Perry, Darling and Thorne, 1959. Consequently, achieving reliable molecular identification for Helicotylenchus species remains a substantial undertaking.

Of the 55 known species in China, two species—H. microlobus and H. membranatus—were not included in the study by Uzma et al. (2015). Firoza and Maqbool (1993) synonymized H. microlobus with H. pseudorobustus, and Uzma et al. (2015) accepted their decision. However, Subbotin et al. (2015) and Mwamula et al. (2020) concluded that these two species could be distinguished from each other based on 28S rRNA D2-D3 expansion segments, and these two species were in two separate clades in this study (Fig. 6). Therefore, we also consider H. microlobus a valid species. Shen (1992), Xie et al. (2004), and Dong et al. (2022) reported H. rotundicauda Sher, 1966 in China. Since H. rotundicauda has been synonymized as H. dihystera (Fortuner 1981; Liu 2005), we transferred their information to H. dihystera. Similar actions were taken on other five species identified in China, i.e., H. bradys Thorne and Malek, 1968, H. glissus Thorne and Malek, 1968, H. nannus Steriner, 1945, H. plumariae Khan & Basir, 1964, and H. talonus Siddiqi, 1972 which were synonymized as H. pseudorobustus, H. dihystera, H. dihystera, H. indicus Siddiqi, 1963, and H. bambesae Elmiligy, 1970, respectively, based on morphological characteristics (Fortuner 1987, 1991; Firoza and Maqbool 1993, 1994). Two other species, H. caribensis Roman, 1965 and H. boringuensis Roman, 1965, were considered species inquirendae by Fortuner (1981). Liu (2005) followed this action and listed them as unconfirmed or species inquirendae, Uzma et al. (2015) also accepted mostly taxonomical status of these species, and we did not include these eight species in this study.

H. dihystera, H. pseudorobustus, H. digonicus, and H. multicinctus are among the most frequently documented species worldwide. Notably, H. pseudorobustus and H. dihystera can parasitize a wide range of crops, impeding their growth, as Subbotin et al. (2015) reported. On the other hand, H. multicinctus is known to induce necrotic lesions on banana roots, ultimately resulting in stunted plant growth, as outlined by Gowen et al. (2005). Although there have been no reported crop losses attributed to these nematodes in China, it is imperative to conduct further examinations to understand their potential threats fully.

Conclusions

This study describes a new species within the genus Helicotylenchus, named H. zengchengensis n. sp., based on its morphological and molecular characteristics. This new species represents the second new species of Helicotylenchus described in China. The identification of this new species contributes to the global diversity of Helicotylenchus, and the DNA sequences obtained will serve as a valuable resource for future molecular diagnostics within the genus. Furthermore, the diagnostic and comprehensive tables included in this report provide information on the geographical distribution and host range of all Helicotylenchus species in China and will significantly facilitate the identification of new record species and research on nematode diversity.

Methods

Nematode population and morphological identification

Soil associated with bamboo was collected from the Zengcheng District, Guangzhou City, Guangdong Province, China (N23.449877° N, 113.847389° E). The nematode was isolated from soil using a modified Barman funnel method, then killed by heating at 65°C for 3 min and fixed with FG fixation (formalin: glycerol: distilled water = 10:1:89) for more than a week, dehydrated using the glycerol-ethanol method and mounted on permanent slides (Xie and Feng 1993). The observation, measurement, and photography of nematodes were performed using a ZEISS Scope A1 light microscope (LM) equipped with a microscopic imaging system and a digital photography camera. For scanning electron microscopy (SEM) studies, the nematodes were observed and photographed under 10 kV using an FEI XL-30-ESEM scanning electron microscope at the Testing Center of South China Agricultural University.

DNA extraction, PCR sequencing, and phylogenetic analyses

DNA was extracted from a single nematode using a proteinase K assay (Mundo-Ocampo et al. 2008). The primers used for amplifying rRNA 28S D2-D3, ITS, and 18S partial sequences and the annealing temperature for PCR reaction were described by Dey ley et al. (1999), Vrain et al. (1992), and Holterman et al. (2006), respectively. PCR amplification was performed in a 40 μL volume of the mixture, including 2 μL DNA template, 2 μL of each primer, 20 μL 2 × Super Taq PCR StarMix (with Dye) (Genestar, Beijing, China), and 14 μL sterile water. The procedure was as follows: pre-denaturation at 94℃ for 3 min, followed by 35 cycles of denaturation at 94℃ for 30 s, annealing at 53℃ for 28S and 55℃ for ITS for 30 s, and 54℃ for 18S rRNA for 1 min, extension at 72℃ for 1 min, and a final extension at 72℃ for 8 min. PCR products were purified using a Gel Extraction Kit (CWBIO, Jiangsu, China). The targeted fragments were cloned into the pMD18-T vector (Takara, Japan), and positive colonies were sequenced by Sangon Biotech Co., Ltd. (Shanghai, China). All obtained sequences were submitted to the GenBank database. Helicotylenchus zengchengensis n. sp. sequences were aligned with other species in the genus Helicotylenchus using BLAST in GenBank under default parameters.

Phylogenetic analysis was used to evaluate the correlations between the new species and other Helicotylenchus species. The phylogenetic trees were constructed by Bayesian inference (BI) using MrBayer 3.2.1 as described by Ronquist et al. (2012). Clustal X was used for sequence alignment with default parameters. Conserved regions were selected with Gblocks 0.91b. The NEX format of aligned sequences was calculated and transformed by PAUP 4.0b4a. The best-fit model was determined by MrModeltest 2.3 based on the Akaike Information Criterion (AIC) with default parameters. Bayesian analysis was initiated with a random starting tree. Four independent Markov Chain Monte Carlo (MCMC) models were used and run for 2 × 10⁶ generations and sampled once every 100 generations. After discarding the first 25% aged samples, the remaining samples were retained to generate a 50% majority rule consensus tree. Posterior probabilities (PP) were given on the clades. For 18S rRNA sequence analysis, 49 sequences from 17 identified and two unidentified species from Helicotylenchus were chosen, with Hoplolaimus galeatus (Cobb, 1913) Thorne, 1935 (Accession: KJ934131) and Hop. seinhorsti Luc, 1958 (Accession: OR288226) as the outgroup. For the ITS sequence analysis, 40 sequences from 13 species from Helicotylenchus were selected, with Rotylenchus pumilus (Perry in Perry, Darling &Thorne, 1959) Sher, 1961 (Accession: JX015436) and Hop. galeatus (Accession: KP303674) as the outgroup. For the 28S rRNA sequences analysis, 46 from 21 identified species and six from unidentified species from Helicotylenchus were selected, with Hop. galeatus (Accession: EU626787) and Hop. seinhorsti (Accession: DQ328752) as the outgroup.