Cryphodera gayae n. sp. (Nematoda: Heteroderidae), from root of Lagerstroemia indica in Korea

Heonil Kang; Jongmin Seo; Garam Han; Donggeun Kim; Insoo Choi

doi:10.21307/jofnem-2019-077

Cryphodera (Colbran, 1966) is a thick-cuticled non-cyst forming nematodes in the group of Heteroderinae. There are seven species of Cryphodera, all parasitic to various species of trees; they are C. brinkmani (Karssen and Van Aelst, 1999), C. coxi, C. nothophagi, C. podocarpi (Wouts, 1973), C. eucalypti (Colbran, 1966), C. kalesari (Bajaj et al., 1989) and C. sinensis (Zhuo et al., 2014). During a survey of plant-parasitic nematodes in Korea, a new species of the non-cyst forming nematode was found from crapemyrtle (Lagerstroemia indica) root. Comparative morphological, morphometric and molecular studies of the nematode revealed that it differed from other Cryphodera species. The new species is described and illustrated herein as Cryphodera gayae n. sp.

Material and methods

Nematode isolation

Cryphodera gayae n. sp. was collected from crapemyrtle roots at Gaya (N35°42′45.2′′, E128°17′30.9), Gyeongsangbuk-do, Korea. Females were hand-picked from roots using forceps. Juveniles (J2) were separated from soil by the modified Baermann funnel method or released from crushed females (Southey, 1986).

Morphological study

Females and juveniles were killed and fixed by addition of 80°C FG 4:1 fixative (Southey, 1986). Nematodes were fixed for at least 24 hr and, then, processed according to the Seinhorst method (Cid Del Prado Vera and Subbotin, 2012). For light microscopic observations, specimens were mounted on Cobb slides and sealed with a paraffin ring and glycerin (Cobb, 1917). Nematodes were observed, measured and photographed with the aid of a compound microscope (BX53, Olympus) equipped with microscope digital camera (DP73, Olympus). For SEM study, nematodes were dehydrated through an ethanol series ranging from 20% through 100%, with specimens left at each step for about 1 day (Cid Del Prado Vera et al., 2012). Subsequently, the specimens were critical point-dried and mounted on studs in suitable position arrangements. Nematodes were coated with gold palladium (SC7620; Polaron) and observed under a scanning electron microscope (Jeol JSM-6390) at 10 kV.

Molecular study

For molecular analysis, DNA was extracted with DNeasy Blood and Tissue Kit (Qiagen Inc., Valencia, CA). Two rRNA fragments, i.e. the LSU D2–D3 and ITS regions, were amplified. Primers for D2–D3 segments amplification were D2A (5′-ACAAGTACCGTGAGGGAAAGTTG-3′) and D3B (5′-TCGGAAGGAACCAGCTACTA-3′). Primers for ITS amplification were TW81 (5′-GTTTCCGTAGGTGAACCTGC-3′) and AB28 (5′-ATATGCTTAAGTTCAGCGGGT-3′). Detailed protocols of polymerase chain reaction (PCR) are as follows: after an initial 5-min denaturation step at 94°C, a 40-cycle amplification (94°C for 1 min, 56°C (D2–D3 segments) to 58°C (ITS region) for 1 min, and 72°C for 2 min) was conducted. The final extension step continued for 10 min at 72°C. In order to confirm the successful amplification of DNA by PCR, electrophoresis was performed using 0.5 × TAE buffer on 1% agarose gel. The PCR product was subsequently purified with a PCR Purification Kit (Qiagen Inc.). The amplicons were cloned in pGEM-T Easy Vector System (Promega), and the resultant plasmid DNA was isolated with a Plasmid Midiprep System (Promega). Both strands of the PCR amplicons were cycle-sequenced with an ABI PRISM BigDye Terminator version 1.1 Cycle Sequencing Kit and electrophoresed in each direction on an ABI Prism ABI 377 Genetic Analyzer (PE Applied Biosystems). The newly obtained sequences were submitted to the GenBank database under accession numbers MN244153 and MN244164.

Phylogenetic study

For phylogenetic analyses, the sequences of C. gayae n. sp. were compared with GenBank nematode sequences using the BLASTn homology search program. The closest sequences were selected for phylogenetic analyses. Outgroup taxa for each data set were chosen according to previous molecular phylogenetic analyses of cyst and non-cyst forming nematodes (Zhuo et al., 2014; Van den Berg et al., 2016; Subbotin et al., 2017, 2018). The newly obtained and published sequences for each gene were aligned using ClustalW (Thompson et al., 1994) with default parameters. Sequence alignments were manually edited using BioEdit (Hall, 1999). The alignment quality was examined visually and optimized manually by adjusting the ambiguous nucleotide positions. Models of base substitution were evaluated using MODELTEST 3.7 (Huelsenbeck and Ronquist, 2001) combined with PAUP 4.0 (Swofford, 2003). The Akaike-supported model, the base frequency, the proportion of invariable sites and the gamma distribution shape parameters and substitution rates in the AIC were then used in phylogenetic analyses. Bayesian analysis was performed to confirm the tree topology for each gene separately using MrBayes 3.2.7 (Ronquist et al., 2012) running the chain for 2.0 × 10⁶ generations and setting the “burn-in” at 2,500. The Markov Chain Monte Carlo method was used within a Bayesian framework to estimate the posterior probabilities of the phylogenetic trees (Larget and Simon, 1999) and generated a 50% majority-rule consensus tree. Posterior probabilities are given on appropriate clades. Trees were visualized using TreeView (Page, 1996).

Results and discussion

Systematics

Cryphodera gayae n. sp.

(Figs. 1–5; Table 1).

Table 1.

Morphometric measurements of Cryphodera gayae n. sp.

1 All measurements are in µm.

Stereo microscopic photos of *Cryphodera gayae* n. sp. A: females attached on crapemyrtle (*Lagerstroemia indica*) root; B: females (scale bars: A = 500 µm, B = 200 µm).

Drawings of *Cryphodera gayae* n. sp. A: female; B: anterior region of J2; C: entire of J2; D: anterior region of female; E: tail region of J2.

Light microscope photos of female of *Cryphodera gayae* n. sp. A: entire of body; B: vulva-anus region (white arrow = anus; black arrow = vulva); C: perineal pattern; E: anterior region (scale bars: A = 50 µm, B–D = 20 µm).

Light microscope photos of second-stage juvenile (J2) of *Cryphodera gayae* n. sp. A: entire body of second-stage juvenile (J2); B: anterior region of J2; C: tail region of J2. Note (*inset*) the middle of hyaline in tail with a constriction; D: head of J2; E: Lateral lines (arrowed) (scale bars: A = 50 µm, B = 20 µm, C = 20 µm, D = 10 µm, E = 10 µm).

SEM photos of second-stage juvenile (J2) of *Cryphodera gayae* n. sp. A: cephalic region; B: lateral field; C: phasmid (scale bars: A = 2 µm, B, C = 1 µm).

Measurements Table 1.

Description

Female

The body is globose, varies in size (513.4–778.3 µm). Cuticle is annulated on entire body (Fig. 3A). Female is pearly white when young and becoming yellowish. Cuticle does not transform to a cyst and is partially covered by some tissue materials (Fig. 1B). Head region is not set off, labial disk is distinct and neck is distinct, usually bent laterally. Stylet is 30.1 µm long. Median bulb is nearly rounded to oval, with distinct valve plates situated centrally (Fig. 3D). Except for the stylet and median bulb, most internal structures are difficult to observe in adult females. Excretory pore is located posterior to metacorpus. Vulva is located sub-terminal to terminal, near the anus. Vulval lips are pronounced with protrusion. Vulva-anus region is slightly concave to flat (Fig. 3B). Mature females contain less than 100 eggs in various stages of development. Perineal patterns are difficult to prepare flat due to a prominent protuberance. Perineal pattern is ovoid with fine, smooth and continuous striae. Vulva is surrounded by circles of striae (Fig. 3C).

Male

Not found.

Second-stage juvenile (J2)

The body is cylindrical, tapering posteriorly, straight or slightly ventrally curved after fixation (Fig. 4A). The lip region is offset, a labial disc with four annuli (Fig. 5A). Stylet is long, cone is straight, and shaft is cylindrical. Stylet knobs are well developed with distinct flat or droop to posterior. Body annuli is 1.5–1.6 µm in width at mid-body and three incisures are visible in lateral position (Fig. 4E). Tail is long conoid, gradually tapering to a sharply pointing terminus. Hyaline terminal section is 38.4 (31.7–47.8) µm long, occupying 49.4 to 62.4% of tail length. Particularly, nearly 65% of J2 specimens of the present population exhibited a distinct constriction around middle of the hyaline region (Fig. 4C).

Diagnosis and relationships

Cryphodera gayae n. sp. has the general characteristics of the genus Cryphodera. Diagnostic characters of C. gayae n. sp. with other species are compiled (Tables 2 and 3). The new species having morphological characters of protruding vulval lips, flat to concave anus-vulva profile, three lateral lines in J2s, appears most similar to C. brinkmani but differs by the shorter stylet length of J2 (26.8–31.3 µm vs 31.6–35.4 µm), stylet knob shape of J2 (flat posteriorly vs concave), egg numbers in mature female (64–69 vs 15–45). Also, J2 of C. brinkmani exhibited a small mucro at the tip of the tail (Vovlas et al., 2013) but C. gayae n. sp. has sharply pointing tip with constriction at the middle of hyaline without mucro-like tip. C. gayae n. sp. differs from C. kalesari by the longer vulva-anus distance (51.9–54.1 µm vs 35 µm), the bigger body size of female (363.1–544.6 µm vs 272–353 µm in length), in J2, the longer tail length (59.6–76.7 µm vs 27–54 µm), longer hyaline length (31.7–47.8 µm vs 18–26 µm) and the shape of tail (sharply pointing tip with constriction at the middle of hyaline vs narrow rounded without constriction). From C. sinensis, the new species differs by the longer J2 tail length (59.6–76.7 µm vs 52.0–65.0 µm) and longer hyaline portion (31.7–47.8 µm vs 24.5–35.0 µm) of J2. C. gayae n. sp. can be distinguished from the remaining four species of Cryphodera, namely, C. eucalypti (Colbran, 1966), C. podocarpi (Wouts, 1973; Luc et al., 1978), C. nothophagi (Wouts, 1973; Luc et al., 1978) and C. coxi (Wouts, 1973; Luc et al., 1978) by the shape of the J2 tail terminus (sharply pointing tip with constriction at the middle of hyaline vs narrow rounded) and the shape of the vulval lips (pronounced protruding vs protruding slightly out of body contour).

Table 2.

Comparative morphometrics (in µm) of female in species of Cryphodera with ranges in brackets.

			Paratype
Stage	Character	Holotype	Range (min.–max.)	Mean ± SD
Female
	n		10
	L (including neck)	462.7	363.1–544.6	471.5 ± 53.8
	L (excluding neck)	380.3	430.6–255.5	367.8 ± 51.4
	W (width)	286.5	166.6–329.1	259.3 ± 46.2
	Neck	82.5	77.2–164.8	103.6 ± 26.9
	L (inc. neck)/W ratio	1.6	1.6–2.2	1.8 ± 0.2
	Stylet length	30.7	28.2–31.9	30.1 ± 1.0
	Anterior end to median bulb	65.0	53.9–93.7	70.3 ± 14.7
	Length of medium bulb	28.3	22.6–31.9	25.9 ± 2.9
	Width of medium bulb	25.2	20.6–29.9	24.3 ± 3.2
	Vulva-nus distance	46.9	35.8–51.6	44.7 ± 4.3
	Vulval slit length	–	51.9–54.1	52.6 ± 1.3
	Excretory pore from anterior end	80.1	72.6–124.5	92.4 ± 17.5
Second stage (J2)
	n		20
	L (body length)	469.1	424.2–525.9	482.8 ± 28.5
	a (L/body diam.)	22.7	18.5–22.7	20.1 ± 1.4
	b (L/esophago-intestinal valve)	3.9	3.4–4.4	3.9 ± 0.3
	b′ (L/end of esophageal)	2.7	2.2–3.0	2.7 ± 0.2
	c (L/tail length)	7.2	6.2–7.4	7.0 ± 0.3
	c′ (tail length/tail diam.)	4.4	3.3–5.0	4.2 ± 0.4
	Stylet length	29.8	26.8–31.3	29.3 ± 1.2
	Tail length	64.8	59.6–76.7	69.4 ± 4.4
	Max. body diam.	20.6	20.3–28.3	24.1 ± 2.5
	Hyaline length	39.0	31.7–47.8	38.4 ± 3.9
	Excretory pore from anterior end	111.93	101.43–132.66	118.6 ± 8.9
Egg
	n		10
	L		92.8–144.2	114.1 ± 15.1
	W		31.4–39.7	36.1 ± 2.8
	L/W ratio		2.9–3.9	3.2 ± 0.3

1 Data referred to Colbran (1966), Wouts (1973), Bajaj et al. (1989), Karssen and Van Aelst (1999), Siddiqi (2000), Sturhan (2010), Zhuo et al. (2014). –, not available.

Table 3.

Comparative morphometrics (in µm) of second-stage juveniles (J2) in species of Cryphodera with ranges in brackets.

	C. gayae n. sp.	C. sinensis	C. eucalypti		C. kalesari	C. brinkmani	C. podocarpi	C. nothophagi	C. coxi
	Present study	Zhuo et al. (2014)	Colbran (1966)	Wouts (1973)	Bajaj et al. (1989)	Karssen and Van Aelst (1999)	Wouts (1973)	Wouts (1973)	Wouts (1973)
Body length (L)	471.5 ± 53.8 (363.1–544.6)	460.9 ± 53.1 (362.9–526.8)	400–636	353 (286–428)	311 ± 21 (272–353)	450 ± 52 (384–544)	393 (300–473)	341 (261–423)	331 (218–455)
Body diameter	259.3 ± 46.2 (166.6–329.1)	240.1 ± 51.6 (172.4–314.0)	201–274	210 (147–290)	205.5 ± 51 (112–288)	238 ± 35 (160–339)	276 (217–335)	226 (148–305)	184 (116–369)
Number of lip annulus	–	–	–	1	2–3	–	2	2 more or less	2 more or less
Vulval slit length	52.6 ± 1.3 (51.9–54.1)	51.3 ± 6.8 (42.8–58.3)	45 (44–49)	–	35	51 ± 3.8 (44–60)	–	–	–
Anus-vulva profile	Flat to concave	Distinctly concave	Flat to concave	Concave	Distinctly concave	Flat to concave	Flat	Flat	Flat

1 DGO, distance of dorsal esophageal gland orifice to base of stylet; Data referred to Colbran (1966), Wouts (1973), Bajaj et al. (1989), Karssen and Van Aelst (1999), Siddiqi (2000), Sturhan (2010), Zhuo et al. (2014). –, not available; ^a Colbran (1966) reported stylet length for J2 of C. eucalypti was 26–35.9 mm. Wouts (1973) mentioned that the minimum 26 mm belonged to J2 of Heterodera. Bajaj et al. (1989) and Karssen and Van Aelst (1999) accepted the point of Wouts (1973). Thus, the value of C. eucalypti described by Colbran (1966) is omitted here; ^b Wouts (1973) described four incisures in the field of C. nothophagi J2. Sturhan (2010) re-examined paratype J2 of C. nothophagi, which revealed three incisures in the field, with the inner incisure occasionally diverging into two. Thus, the point of Sturhan (2010) is accepted here.

Molecular profiles and phylogenetic status

The sequenced LSU D2–D3 segments and ITS region are 792 and 1,028 bp, respectively. A BLASTn search of C. gayae n. sp. on the LSU D2–D3 segments revealed high-scoring matches with some Cryphodera species, the most similar to C. sinensis (GenBank accession number JX566455), from ramie in Hunan Province in China. The identity of sequence was 95% (750/789), with four insertions/deletions (0.1%). A BLASTn search of C. gayae n. sp. on the ITS region also revealed similarities with some Cryphodera species, but the identities were about 85%. The highest match was C. brinkmaini (JQ965678), with only 88% identity (618/703) and 23 insertions/deletions (3%). Compared with C. sinensis (JX566457), C. gayae n. sp has 83% identity (892/1,075) and 85 insertions/deletions (7%).

The molecular phylogenetic relationships of the new species are shown in Figures 6 and 7. Figure 6 represents a phylogenetic tree based on LSU D2–D3 segments. The average nucleotide composition is as follows: 22.34% A, 22.47% C, 29.58% G and 25.61% T. Using Scutellonema brachyurus as the outgroup taxa, the molecular phylogeny strongly supports monophyly of Cryphodera (100% posterior probability). Figure 7 represents a phylogenetic tree based on ITS region. The average nucleotide composition is as follows: 21.11% A, 22.91% C, 27.17% G and 28.81% T. Using Hoplolaimus columbus as outgroup taxa, all Cryphodera sequences reside within a well-supported monophyletic clade with 100% support. The sequence of C. gayae n. sp. clade is in separate position to C. sinensis founded from ramie (Zhuo et al., 2014).

Phylogenetic relationships within population and species of *Heteroderidae*. Bayesian 50% majority-rule consensus tree from two runs as inferred from the analysis of the D2–D3 of 28 S rDNA gene sequences under the GTR + I + G model (1nL = 15776.80; AIC = 31899.59; freqA = 0.2234; freqC = 0.2247; freqG = 0.2958; freqT = 0.2561; R(a) = 0.3202; R(b) = 2.082; R(c) = 1.008; R(d) = 0.2953; R(e) = 4.3966; R(f) = 1; Pinva = 0.3467; Shape = 0.7997). Posterior probability values more than 70% are given in appropriate clades. Newly sequenced sample is indicated by red color and bold font.

Type locality and habitat

Cryphodera gayae n. sp. was collected from roots of crapemyrtle (Lagerstroemia indica L.), Gaya district (N35°42′45.2″, E128°17′30.9), Gyeongsangbuk-do, Korea in 2019. The soil type is sandy loam, and the local climate is marine west coast of Köppen climate classification.

Type specimens

Holotype (female and J2): isolated from roots from type locality and habitat. Slides T-721t and T-722t deposited in the United States Department of Agriculture Nematode Collection (USDANC), Beltsville, Maryland. Paratype (female and J2): same data and repository as holotype. Slides T-7228p and T-7229p.

Etymology

The specific epithet refers to the Gaya dynasty before 400 AD in ancient history of Korea. The species habitat is the geographical capital region of Gaya. Gaya was a confederacy of territorial polities nobility in the Nakdong river basin of southern Korea.

Key to the species of Cryphodera, based on second-stage juveniles:

1. — Three lip annules (lateral)2

— Four to five lip annules (lateral)4

2. — Stylet ≤ 29 μm, knobs anteriorly flatC. kalesari

— Stylet > 29 μm3

3. — Tail of J2 with narrow roundedC. eucalypti

— Tail of J2 with point, mucro-like tipC. sinensis

4. — Four lip annules5

— Five lip annulesC. podocarpi

5. — Three lateral lines near mid-body6

— Four lateral lines near mid-bodyC. nothophagi

6. — Tail length < 60 μm, tail tip narrow roundedC. coxi

— Tail length ≥ 60 μm, tail tip pointed7

7. — Tail of J2 with mucro-like tip; stylet = 33.3 μm, DGO = 5.8C. brinkmani

— Tail of J2 with constriction at the middle of hyaline; style L = 29.8,DGO = 4.6C. gayae n. sp.

Acknowledgments

This work was carried out with the support of “Cooperative Research Program for Agriculture Science and Technology Department (Project No. PJ0134282019)” Rural Development Administration, Republic of Korea.

	C. gayae n. sp.	C. sinensis	C. eucalypti		C. kalesari	C. brinkmani	C. podocarpi	C. nothophagi	C. coxi
	Present study	Zhuo et al. (2014)	Colbran (1966)	Wouts (1973)	Bajaj et al. (1989)	Karssen and Van Aelst (1999)	Wouts (1973)	Wouts (1973)	Wouts (1973)
Body length (L)	482.8 ± 28.5 (424.2–525.9)	435.2 ± 25.4 (388.8–474.5)	379–461	428 ± 6	383 ± 19 (353–424)	393 ± 23 (450–541)	525 ± 5	450 ± 4	457 ± 4
a = L/max. width	20.1 ± 1.4 (18.5–22.7)	22.6 ± 1.5 (19.4–25.9)	22.0–26.0	24.8 ± 0.6	22 ± 1.5 (18–26)	23.2 ± 1.3 (20.3–24.6)	27.2 ± 0.5	24.5 ± 0.3	25.3 ± 0.4
b = L/esophageal length	3.9 ± 0.3 (3.4–4.4)	3.9 ± 0.2 (3.7–4.4)	3.5-4.8	3.7 ± 0.06	3.84 ± 0.38 (3.2–5.0)	–	3.8 ± 0.06	3.7 ± 0.05	3.7 ± 0.04
c = L/tail length	7.0 ± 0.3 (6.2–7.4)	7.8 ± 0.5 (6.4–8.6)	7.9–9.8	8.3 ± 0.15	8.4 ± 1.2 (7–13)	7.1 ± 0.4 (6.4–7.8)	7.7 ± 0.07	8.2 ± 0.08	8.5 ± 0.08
Number of lip annuli	4	3	3	3	3	4	5	4	4
Stylet length	29.3 ± 1.2 (26.8–31.3)	29.1 ± 0.8 (28.0–31.0)	– ^a	32.9 ± 0.3 (31–35)	26.5 ± 1 (24–29)	33.3 ± 1.0 (31.6–35.4)	38.7 ± 0.4	33.9 ± 0.2	34.4 ± 0.2
Stylet knobs	Flat posteriorly	Project distinctly anteriorly	Concave	Flat or pointing slightly anteriorly	Flat to concave	Concave	Pointing distinctly anteriorly	Flat anteriorly	Pointing slightly anteriorly
DGO	4.6 ± 0.9 (3.3–6.1)	5.4 ± 0.7 (4.5–6.5)	4.3–5.0	5.5 ± 0.17	3–4	5.8 ± 0.6 (4.4–7.0)	6.0 ± 0.26	5.6 ± 0.17	6.1 ± 0.28
Number of lateral line	3	3	3	3	3	3	3	3 (occasionally 4)^b	3
Tail length	69.4 ± 4.4 (59.6–76.7)	56.0 ± 3.2 (52.0–65.0)	–	46.5 ± 6 (27–54)	46.5 ± 6 (27–54)	69.7 ± 4.4 (61.3–76.5)	68.9 ± 0.1	55.3 ± 0.8	53.7 ± 0.8
Hyaline portion of tail	38.4 ± 3.9 (31.7–47.8)	28.3 ± 2.3 (24.5–35.0)	–	21.5 ± 2.5 (18–26)	21.5 ± 2.5 (18–26)	37.2 ± 3.6 (30.3–42.3)	36.0 ± 0.1	30.1 ± 0.7	30.6 ± 0.5
Tail terminus	Sharp point with constriction at the middle of hyaline	Point with mucro-like tip	Buntly rounded	Narrow rounded	Narrow rounded	Point with mucro-like tip	Narrow rounded	Narrow rounded	Narrow rounded

Cryphodera gayae n. sp. (Nematoda: Heteroderidae), from root of Lagerstroemia indica in Korea

Full Article

Material and methods

Nematode isolation

Morphological study

Molecular study

Phylogenetic study

Results and discussion

Table 1.

Figure 1:

Figure 2:

Figure 3:

Figure 4:

Figure 5: