A new stunt nematode, Geocenamus chengi n. sp. (Nematoda: Merliniinae) in the rhizosphere of tea (Camellia sinensis ) from Zhejiang Province, China
Data publikacji: 16 kwi 2020
Zakres stron: 1 - 13
Otrzymano: 02 lut 2020
DOI: https://doi.org/10.21307/jofnem-2020-042
Słowa kluczowe
© 2020 Munawar Maria et al., published by Sciendo.
This work is licensed under the Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
The tea plant is native to China; this country has the highest tea production and consumption rate in the world. More than 100,000 hectares of tea acreage are located in seven Chinese provinces: Yunnan, Sichuan, Fujian, Hubei, Zhejiang, Guizhou, and Anhui (Yao and Chen, 2012). In an attempt to investigate the nematode biodiversity associated with the tea plantations of Hangzhou, Zhejiang Province, a population of stunt nematode of subfamily Merliniinae was detected. This family comprises migratory ectoparasites of roots; merlinid nematodes are root feeders and can subsist on a variety of host plants (Siddiqi, 2000). The detected population of stunt nematode was examined carefully, and the morphological characterization indicated that this population belongs to the genus
Figure 1:
Line drawings of
Figure 2:
Light photomicrographs of
Figure 3:
Light photomicrographs of
Figure 4:
Scanning electron microscopy of
Morphometric data for
| Holotype | Paratype | ||
|---|---|---|---|
| Female | Male | ||
|
|
20 | 10 | |
| Body length | 884.8 | 879.0 ± 59.6 (783–997) | 822.8 ± 34.2 (772–892) |
| a | 33.3 | 33.3 ± 2.6 (28–39) | 32.9 ± 1.2 (31–34) |
| b | 6.0 | 6.1 ± 0.3 (5.5–6.6) | 6.1 ± 0.2 (5.8–6.6) |
| c | 10.4 | 10.5 ± 0.6 (9.2–11.7) | 9.7 ± 0.6 (8.7–10.3) |
| c' | 4.7 | 4.6 ± 0.4 (4.2–5.5) | 4.8 ± 0.3 (4.5–5.2) |
| V | 52.8 | 52.5 ± 1.6 (49.5–55.7) | 39.4 ± 5.4 (33.1–47.4) |
| Lip height | 7.5 | 7.6 ± 2.4 (7.0–8.0) | 7.6 ± 0.3 (7.0–8.0) |
| Lip width | 3.7 | 3.8 ± 0.3 (3.4–4.5) | 3.7 ± 0.3 (3.2–4.0) |
| Stylet length | 22.3 | 22.2 ± 0.6 (21–23) | 21.5 ± 0.7 (20–23) |
| DGO distance from stylet knobs | 2.0 | 2.0 ± 0.1 (1.7–2.2) | 2.1 ± 0.1 (1.9–2.3) |
| Excretory pore from anterior end | 122.8 | 120.6 ± 5.2 (109–126) | 108.3 ± 5.2 (101–116) |
| Hemizonid from anterior end | 118.4 | 116.7 ± 5.1 (104–123) | 103.0 ± 6.7 (94–113) |
| Pharynx length | 148.4 | 145 ± 5.7 (130–152) | 134 ± 3.6 (127–139) |
| Maximum body diam. | 26.6 | 26.6 ± 2.5 (22–33) | 25.0 ± 1.0 (23–27) |
| Vulval body diam. | 25.4 | 26.6 ± 2.3 (23–32) | − |
| Anal/cloacal body diam. | 18.3 | 18.1 ± 1.2 (16–21) | 17.7 ± 0.9 (16–19) |
| Tail length | 85.2 | 83.8 ± 5.4 (76–92) | 85.2 ± 5.4 (78–93) |
| Hyaline tail part length | 25.2 | 22.4 ± 2.2 (19–25) | 21.3 ± 1.9 (18–24) |
| Phasmid position from anus/cloaca | 12.4 | 15.5 ± 4.1 (10–19) | 24.6 ± 3.9 (19–29) |
| Phasmid % of tail | 14.6 | 17.4 ± 4.0 (12–20) | 29.5 ± 4.0 (24–35) |
| Spicule length | − | 23.4 ± 1.9 (22–25) | |
| Gubernaculum | − | 8.0 ± 0.7 (7–9) | |
The status of genera in subfamily Merliniinae (Siddiqi, 1971) has been discussed by several nematologists, namely Andrássy (1977), Hooper (1978), Fortuner and Luc (1987), Siddiqi (1986, 2000), and Brzeski (1991). However, based on the recent classification (Geraert, 2011), the subfamily Merliniinae contains
Currently, the genus
Nematodes were extracted from soil and root samples using the modified Cobb sieving and flotation-centrifugation method (Jenkins, 1964). For morphometric studies, nematodes were killed and fixed in hot formalin (4% with 1% glycerol) and processed in glycerin (Seinhorst, 1959). The measurements and light micrographs of nematodes were made with a Nikon Eclipse Ni-U 931845 compound microscope. For the SEM examination, the nematodes were fixed in a mixture of 2.5% paraformaldehyde and 2.5% glutaraldehyde, washed three times in 0.1 M cacodylate buffer, post-fixed in 1% osmium tetroxide, dehydrated in a series of ethanol solutions, and critical point-dried with CO2. After mounting on stubs, the samples were coated with gold at 6 to 10 nanometer thickness, and the micrographs were made at 3 to 5 kV operating system (Maria et al., 2018).
DNA was extracted by transferring individual nematodes into an Eppendorf tube containing 16 μL ddH2O. Nematodes were crushed using a sterilized pipette tip, and the tubes were centrifuged at 12,000 rpm for 1 min and frozen at −68°C for at least 30 min. Tubes were heated to 85°C for 2 min, and then, 2 μL proteinase K was added in PCR buffer solution. The tubes were incubated at 56°C for 1 to 2 hr and at 95°C for 10 min. After incubation, these tubes were cooled to 4°C and used for conducting PCR analyses (Zheng et al., 2003). Several sets of primers (synthesized by Invitrogen, Shanghai, China) were used in the PCR analyses to amplify the partial 18 S, ITS region and D2-D3 expansion domains of 28 S of rDNA. Primers for amplification of partial 18 S were 18s900-18s1713 (Olson et al., 2017). Primers for amplification of ITS were TW81-AB28 (Joyce et al., 1994). The primers for amplification of D2-D3 of 28 S were D2A and D3B (De Ley et al., 1999). PCR conditions were as described by Ye et al. (2007) and Powers et al. (2010). PCR products were evaluated on 1% agarose gels stained with ethidium bromide. PCR products of sufficiently high quality were sent for sequencing by Invitrogen (Shanghai, China).
The newly obtained sequences were deposited into the GenBank database, and accessions were in the phylogenetic trees. The DNA sequences were compared with those of the other merlinids and related nematodes available at the GenBank sequence database using the BLAST homology search program. Outgroup taxa for the data set were chosen according to previously published data (Handoo et al., 2014; Nguyen et al., 2019). Multiple alignments of the different sequences were made using the Q-INS-i algorithm of MAFFT v. 7.205 (Katoh and Standley, 2013). The best-fit model of DNA evolution was obtained using jModelTest V.2.1.7 (Darriba et al., 2012) with the Akaike information criterion (AIC). The best-fit model, the base frequency, the proportion of invariable sites, and the gamma distribution shape parameters and substitution rates in the AIC were then given to MrBayes for the phylogenetic analyses. Transitional model and gamma-shaped distribution (TIM3ef + G) were used for the 18 S; unlinked general time-reversible model with invariable sites and a gamma-shaped distribution (GTR + I + G) was used for D2-D3 expansion domains of 28 S, and transitional model with invariable sites and a gamma-shaped distribution ( TIM2 + I + G) for ITS. Bayesian analysis was performed to confirm the tree topology for each gene separately using MrBayes 3.1.0 (Huelsenbeck and Ronquist, 2001) 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 10% 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 (
After fixation, the body is ventrally curved or C shaped. There are six incisures on lateral field, areolation at mid-body and tail region is observed in the majority of individuals. Body annuli are clearly defined and divided into blocks (seen under SEM). Labial region is dome shaped and slightly offset from the rest of the body having four to five annuli. Irregular rounded rectangular labial disc is observed surrounded by the dorsal, ventral sectors, and amphidial apertures. The labial framework is not sclerotized. Stylet is well-developed with rounded knobs. The dorsal gland orifice is located 2.0 μm posterior to stylet knobs. Median bulb is oval with bean-shaped central valve plates; deirid is absent. Isthmus is slender, surrounded by a nerve ring; pharyngeal basal bulb is saccate and abutting intestine. Cardia is indistinct and conoid rounded. Excretory pore is located at the anterior region of basal pharyngeal bulb. Hemizonid is 2-3 annuli long, anterior to excretory pore; vulva is a transverse slit, vulval lips are elongated and ellipsoidal with epiptygma (SEM), and vagina is “v” shaped comprising less than half of the corresponding diameter; spermatheca is rounded filled with rounded sperm cells; ovaries are outstretched with a single row of oocytes. The tail is annulated, elongated, and conical with a terminal hyaline region, which comprises 21 to 33% of the tail length, ending as bluntly pointed tip. Phasmid is small and pore like, located 10 to 19% posterior from anus.
Body habitus, cuticle and anterior region of males are similar to females. Gonad is located on the right side of the intestine and outstretched. Spicule is 22 to 25 μm long with truncated head and having an abrupt depression just below the head on the dorsal side, followed by a curve that tapers till the distal end of the spicule, and distal tips are bluntly pointed; gubernaculum is saucer shaped; cloacal lips are not protuberant, and two posterior hypoptygmata can be seen under SEM. Bursa crenate covers the tail until the hyaline tail region. Tail shape is similar to that of female, and terminal hyaline region comprises 20 to 28% of the tail length. Phasmid is small and located 19 to 29% posterior from cloaca.
This population was detected in the rhizosphere of
Holotype female and 16 female and 8 male paratypes (slide numbers ZJU-31-01-ZJU-31-10) were deposited in the nematode collection of Zhejiang University, Hangzhou, China. Additional 10 slides having plenty of male and female were also stored in the same collection. Also, 4 females and 2 male paratypes (slide numbers T-7378p, T-7379p) were deposited at USDA nematode collection, Beltsville, Maryland, USA.
The species is named after late Professor Hurui Cheng, one of the famous plant nematologists in China, for his extraordinary contribution to the nematode taxonomy from China.
Based on the similar tail morphology,
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In the 18 S gene analysis (Fig. 5), the
Figure 5:
Phylogenetic relationships of
Figure 6:
Phylogenetic relationships of
Figure 7:
Phylogenetic relationships of
In all the phylogenetic analyses,
The classification presented by Siddiqi (2000) and Andrássy (2007) distinguished
In our phylogenetic analysis,
The present study does not address any taxonomic revisions or higher classification; however, it describes a new