Tylenchus zeae, Kantor & Handoo & Subbotin & Mowery & Hult & Rogers & Skantar, 2023

Kantor, Mihail R., Handoo, Zafar A., Subbotin, Sergei A., Mowery, Joseph D., Hult, Maria N., Rogers, Stephen & Skantar, Andrea M., 2023, Molecular and morphological characterization of Tylenchus zeae n. sp. (Nematoda: Tylenchida) from Corn (Zea mays) in South Carolina, Journal of Nematology 55 (1), pp. 1-13 : 3-9

Tylenchus zeae n. sp. ( Figs. 1-5 ).

Measurements

Measurements are given in Table 1 .

Description

Female: The female body is vermiform, assuming arcuate C-shaped form when killed by gentle heat. Cuticle is strongly striated. The lip region is striated, and the head almost continues but is distinctly offset and bearing five or six annules. In SEM ( Fig. 1 ) four to six cephalic sensilla were noticed, represented by small pits at the rounded corners of the labial plate. The oral plate is small and round. The amphideal opening is large and pit-like, confined to the labial plate, extending three or four annules beyond the labial plate. The stylet is heavy and well developed, between 20 and 22 µm long. The conus is half of the stylet length. Basal knobs are rounded, well developed, and sloping posteriorly. The median bulb is large and oval, with an elongated basal bulb. The Excretory pore is visible and the excretory duct is heavily sclerotized. Four wide lines in the lateral fields are characteristic with crenate outer margins ( Figs. 2D ; 3C ) and outer bands areolated with areolation mostly not joining. The female gonad is long and outstretched, almost reaching to the pharynx area with round to oval spermatheca filled with large round sperms. Vulva lips were not protruding but occasionally noticed in a few specimens slightly protruding. The vagina is about one third of the body width at vulva. The post vulval uterine sac is more than half of the vulval diameter. The tail is slender, almost cylindrical, regularly tapering, and arcuate with its terminus finely to bluntly rounded. Tail annulation is prominent, extending to the tail terminus.

Male: The male is similar to females in general body characteristics, except for the reproductive organs. Spicules are long, curved ventrally, and gubernaculum is small. Bursa adanal is short. Tail is an elongate conoid, with a tail terminus bent ventrally, and finely rounded.

Diagnosis and relationships

Tylenchus zeae n. sp. females are similar to Tylenchus rex Andrassy, 1979 from which they differ by having a slightly different body shape (vermiform, assuming arcuate C shaped vs. body almost straight, hardly curved), shorter body length of 830 (765–895) µm vs. 1,007 (960–1009) µm, a basal bulb that is long and moderately developed, elongated vs. fairly small, spoon-shaped, and a shorter distance from anterior end to excretory pore (103 vs. 130 µm), as well as the shorter tail length (112–127 µm vs. 127–160 µm) with tail not showing two slightly visible breaks and tail terminus not pointed vs. rounded when compared to the T. rex population reported by Andrassy (1979). The a, b, c, and V % ( Table 2 ) values are within the range reported by Brzeski (1996). Males of T. zeae n. sp. have longer spicules, 23 to 27 µm vs. 20 to 23 µm.

Tylenchus zeae n. sp. females are close to T. sherianus Andrassy, 1981 , although they differ from the latter by the shape of the median bulb, which is oval in T. zeae n. sp. compared with a more rhombus shape in T. sherianus ; longer stylet length (20–22 μm vs. 19–20 μm); a higher a value (28–42 vs. 25–28); and a b value that is also slightly larger (6–8 vs. 5–7); and a slightly lower V % (61–65% vs. 65–68%). The tail length is within the range (112–127 vs 100–116 μm). The shape of the excretory duct is different in T. zeae n. sp. when compared to T. sherianus , in which the duct is doubled curved, forming an S shape, whereas in T. zeae n. sp. it is heavily sclerotized and straight. The lateral field has four lines that are characteristic with crenate outer margins and outer bands areolated with areolation mostly not joining vs. a lateral field with four narrow, smooth lines with no crenated margins or areolations in T. sherianus . T. zeae n. sp. have a bluntly rounded tail terminus, vs. a pointed terminus in T. sherianus . Males of T. zeae n. sp. have a shorter tail (122–135 μm vs. 155 μm) than given for T. sherianus in the original description and it is sharply pointed. The gubernaculum is also slightly shorter in T. zeae n. sp. (5–6 μm vs. 8 μm).

The other two species that T. zeae n. sp. comes close to are Tylenchus davainei Bastian, 1865 and Tylenchus arcuatus Siddiqi, 1963 . Tylenchus zeae n. sp. differs from Tylenchus arcuatus by having a longer stylet (20–22 μm vs. 15–17.5 μm). The a value is much higher in T. zeae n. sp. (28–42 vs. 23–29 in T. arcuatus ), the b value for T. zeae n. sp. is 7.0 (6–8), whereas for T. arcuatus it is 6.0 (4.9–6.0). T. zeae n. sp. have a smaller V % (61–66% vs. 64–71%) and a longer tail (112– 160 μm vs. 80–137 μm) that is finely rounded vs. robust, with a hooked, sharply broken, pointed tip. Females differ from T. davainei by having a longer stylet (20.0– 22.0 μm vs. 15.8–17.6 μm) with rounded stylet knobs vs. anteriorly directed knobs, and a slightly shorter body length (765.0–895.0 μm vs. 863.0-1182.0 μm). Males of T. zeae n. sp. have a larger a value (35.0–39.0 vs. 28.9- 35.2) than T. davainei .

Type host and locality

T. zeae n. sp. is associated with roots and soil of corn ( Zea mays ) in Pickens County, South Carolina . The Global Positioning System coordinates are 35.00341 N, 82.65306 W.

Type material

HOlOtype (Female): Slide T- 766t, deposited in the United States Department of Agriculture Nematode Collection, Beltsville, MD, USA.

Paratypes (Females and Males): Same data and repository as holotype. Slides T-7629p–T-7635p.

ZOObank ID: urn:lsid:zoobank.org:act:430F0F46-8163-4636-8FC3-97D90A0B0519

Etymology

The species name is derived from Zea mays , the host of this species.

Molecular phylogenetic analysis

Amplification and sequencing of 18S rRNA resulted in a 1605 bp fragment from PCR with primers SSU_F_04 and SSU_R_81 from one individual. For three other nematodes, primers 18 S1.2 and 18Sr2b were used to generate fragments of 555 to 575 bp. Because the longer fragment contained more variable sites for comparison, that sequence was used for subsequent phylogenetic analysis. BlastN comparison to sequences in GenBank resulted in the highest similarity to Filenchus vulgaris (KX156307), differing at 32 bp (2.24%) and to Tylenchus arcucatus (EU306348), differing at 36 bp (2.24%). For the 18S rRNA gene, 59 sequences from 44 taxa were used to construct a 1598 bp alignment used for Bayesian inference. In the 18S tree ( Fig. 4 ), T. zeae n. sp. and two sequences from T. arctuatus grouped together but were unresolved. All three of these sequences appeared within a larger clade that included a third T. arcuatus sequence, an unspecified Tylenchus sp. , several Filenchus spp. , and a subclade containing T. naranensis and T. davanei .

For 28S rRNA, amplification from five individuals resulted in a 738 bp fragment. Sequences obtained from PCR of four of five specimens were of insufficient quality to generate independent contigs, although partial reads did match the full-length D2-D3 obtained from the high-quality sequence. The representative sequence used for subsequent analysis was 79.77% similar to one from an undescribed Tylenchus sp. CD207 from palm soil in Florida (JX291130), differing at 140 bp. The highest pairwise similarity among all sequences available in GenBank (82.95%) was to HelicOtylenchus Oleae (MF287653). For 28S phylogenetic analysis, 69 Tylenchidae sequences were used to construct an 822 bp alignment. In the inferred 28S MrBayes tree ( Fig. 5 ), the Tylenchus zeae n. sp. grouped with an undescribed Tylenchidae sp. (JX291130) with strong support (PP = 1.00). However, the genus did not form a monophyletic group, with T. arcuatus and other Tylenchus populations forming clades with Filenchus species and others with Litylenchus crenatae. This finding is consistent with the polyphyletic groupings observed by others ( Atighi et al., 2013; Qing et al., 2017; Qing and Bert, 2019; Bai et al., 2020).

For mitochondrial COI, fragments of 416 bp were amplified from two nematodes, differing from each other at a single nucleotide. Sequence similarity was highest (90.38%) with Tylenchus arcuatus from China (MN577620), differing at 40 bp (9.6%). The COI gene tree contained 18 sequences from fewer taxa but with strong branch support for individual clades; the SC population appeared as a sister group to T. arcuatus and was clearly resolved from Filenchus vulgaris ( Fig. 6 ). A relative lack of available COI gene sequences prevented further cross-comparison of the T. zeae n. sp. position relative to most other species present within the 18S rRNA gene tree. Tylenchus zeae differs from its nearest neighbor in the 18S tree, T. arcuatus , at 2.5% pairwise difference and at 9.9% in the COI. In comparison, F. vulgaris , which is also poorly resolved from T. arcuatus in the18S tree, differs at 0.6% in this gene and at 16.1% in COI. Although not perfect parallel comparisons, they do give some measure of the relative sequence diversity in 18S and COI, as reflected in the separate species.

Figures

Figure 1: Scanning electron micrograph (SEM) images of TylenChus zeae n. sp. A: Female specimen, anterior end, arrow pointing toward the excretory pore; B: Female specimen, head; C: Female specimen, face view; D: Lateral field (midbody); E: Female specimen, anal opening; F: Female specimen, vulval opening; G: Male specimen, spicule; H: Female specimen, arrow showing the anal opening; I: Female specimen, tail; J: Male specimen, posterior end.

Figure 2: Photomicrographs of TylenChus zeae n. sp. males and females. A–B: Anterior end with arrows pointing toward the excretory pore; C: Excretory pore; D: Areolated lateral field; E: Entire female body; F: Female basal bulb; G: Female gonad; H–I: female posterior end with arrow pointing the anal area (H); J: Female vulva region with arrow pointing toward the spermatheca; K: Male spicule.

Figure 3: Line drawings of TylenChus zeae n. sp. A: Female pharyngeal region; B: Female lip region showing stylet; C: Areolated lateral field; D: Male spicule, gubernaculum, and bursa. E: Vulval region showing vulva, uterus, and spermatheca; F–G: female tails.

Figure 4: Phylogenetic relationships of TylenChus zeae n. sp. with other select Tylenchidae, as inferred from a 1585 bp alignment of 18S rRNA sequences, according to the GTR + I + G model of nucleotide substitution and incorporated into MrBayes (MB) as described. A 50% majority rule consensus tree was generated with posterior probabilities (PP) shown on appropriate branches, with AphelenChOides besseyi as the outgroup. New sequences are indicated in bold.

Figure 5: Phylogenetic relationships of TylenChus zeae n. sp. with other select Tylenchidae, as inferred from an 822 bp alignment of 28S rRNA sequences, according to the GTR + I + G model of nucleotide substitution and incorporated into MrBayes (MB) as described. A 50% majority rule consensus tree was generated with posterior probabilities (PP) shown on appropriate branches, with BursaphelenChus muCrOnatus as the outgroup. New sequences are indicated in bold.

Figure 6: Phylogenetic relationships of TylenChus zeae n. sp. with other select Tylenchidae, as inferred from a 418 bp alignment of mitochondrial COI sequences, according to the GTR + I + G model of nucleotide substitution and incorporated into MrBayes (MB) as described. A 50% majority rule consensus tree was generated with posterior probabilities (PP) shown on appropriate branches, with BursaphelenChus COniCaudatus as the outgroup. New sequences are indicated in bold.

Tables

Table 1. Morphometrics of TylenCHUS Zeae n. sp. All measurements are in μm and in the form: mean ± standard deviation (range).

Character	Holotype	Female (n = 9)	Male (n = 4)
L	830	830.0 ± 39.3 (765.0–895.0)	813.0 ± 45 (775.0–885.0)
Stylet	20	20.0 ± 0.7 (20.0–22.0)	20.0 (20.0–20.0)
MBW	27	25.0±2.58 (20.0–30.0)	22.0 (20.0–25.0)
Ant. end to exc. pore distance	102	103.0 ± 6.0 (92.0–115.0)	—
Ant. end to esophago-intestinal valve	122	123.0 ± 5.5 (115.0–130.0)	123.0 ± 2.0 (120.0–125.0)
Tail	112	121.0 ± 5.9 (112.0–127.0)	127.0 ± 5.0 (122.0–135.0)
a	31	33.0 ± 3.7 (28.0–42.0)	37.0 ± 2.0 (35.0–39.0)
b	7	7.0 ± 0.5 (6.0–8.0)	7.0 ± 0.4 (6.0–7.0)
c	7	7.0	6.0 ± 0.3 (6.0–7.0)
V %	63	63.0 ± 1.2 (61–65%)	—
Anal body width	15	15 ± 1.28 (12.0–17.0)	13.0 (12.0–14.0)
Spicules	—	—	21.0 ± 1.0 (20.0–23.0)
Gubernaculum	—	—	6.0 ± 0.5 (5.0–6.0)

L, body length; MBW, maximum body width.