Updated description of Paratylenchus lepidus Raski 1975 and P . minor Sharma, Sharma and Khan, 1986 by integrating molecular and ultra-structural observations
Article Category: Arts & Humanities
Publié en ligne: 17 sept. 2019
Pages: 1 - 13
Reçu: 28 juin 2019
DOI: https://doi.org/10.21307/jofnem-2019-056
© 2019 Munawar Maria et al., published by Sciendo
This work is licensed under the Creative Commons Attribution 4.0 International License.
The species of pin nematodes are commonly distributed in China, more than 30 species have been reported from 21 provinces (Maria et al., 2018a). The host range varies from horticultural to agronomic crops as well as ornamental and forestry plants (Raski, 1991). The species of pin nematodes were divided into two genera, i.e.
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 to 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-nm thickness and the micrographs were made at 3 to 5 kV operating system (Maria et al., 2018c).
DNA was extracted by transferring individual nematodes into an Eppendorf tube containing 16 μ L ddH2O. Nematodes were crushed using a sterilized pipette tip, 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, then 2 μ L proteinase K was added and PCR buffer solution. The tubes were incubated at 56°C for 1 to 2 hr, followed by 10 min at 95°C. After incubation, these tubes were cooled to 4°C and used for conducting PCR (Zheng et al., 2003). Several sets of primers (synthesized by Invitrogen, Shanghai, China) were used in the PCR analyses to amplify the partial 18S, ITS region and D2-D3 of 28S of rDNA. Primers for amplification of partial 18S were 18s39F-18s977R and 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 28S 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 sequences were deposited into the GenBank database. DNA sequences were aligned by MEGA7 (Kumar et al., 2016) using default settings. The DNA sequences were compared with those of the other pin nematode species available at the GenBank sequence database using the BLAST homology search program. The model of base substitution was evaluated using MODELTEST (Posada and Crandall, 1998; Huelsenbeck and Ronquist, 2001). The Akaike-supported model, the base frequencies, the proportion of invariable sites, and the gamma distribution shape parameters and substitution rates were used in phylogenetic analyses. Bayesian analysis was performed to confirm the tree topology for each gene separately using MrBayes 3.1.0 (Huelsenbeck and Ronquist, 2001) running the chain for 1 × 106 generations and setting the “burnin” at 2,500. The Markov Chain Monte Carlo (MCMC) method was used within a Bayesian framework to estimate the posterior probabilities of the phylogenetic trees (Larget and Simon, 1999) using 50% majority rule.
Systematics
Figure 1:
Light photomicrographs of
Figure 2:
Scanning electron microscopy of
Measurements of the females of
Morphometric data for
| Type | Additional type pop. | ||||
|---|---|---|---|---|---|
| This study | Raski (1975b) | Raski (1975b) | Chen et al. (2007) | Yu et al. (2014) | |
|
|
|
|
a |
|
|
| Characters/ratios | Taizhou | Sri Lanka | Sri Lanka | Taiwan | Hunan |
| n | 23 | 21 | 13 | 26 | |
| Body length | 324.2 ± 21.0 (288.4–359) | 330 (280–400) | 320 (290–370) | 270–400 | 260–420 |
| a | 27.4 ± 2.5 (22.2–31.6) | 25 (23–31) | 27 (23–31) | 19.4–31 | 13.1–23.1 |
| b | 4.0 ± 0.3 (3.5–4.4) | 4.0 (3.4–4.0) | 3.8 (3.5–4.6) | 3.2–4.6 | 3.3–5.1 |
| c | 10.4 ± 0.6 (9.2–11.6) | 14 (11–16) | – | 11–16 | 5.3–10.3 |
| c' | 3.8 ± 0.2 (3.3–4.3) | – | – | – | |
| V | 81.4 ± 0.9 (80.0–83.3) | 82 (80–84) | 82 (80–83) | 78.5–84 | 78–84 |
| Lip height | 3.1 ± 0.2 (2.6–3.4) | – | – | – | – |
| Lip width | 5.5 ± 0.3 (5.1–6.2) | – | – | – | – |
| Stylet | 27 ± 1.0 (24.8–28.7) | 25 (22–27) | 25 (23–28) | 21.3–27 | 13.7–27.9 |
| Stylet percentage | 8.3 ± 0.4 (7.6–9.2) | – | – | – | – |
| Median bulb Length | 18.0 ± 1.4 (15.2–19.9) | – | – | – | – |
| Median bulb width | 6.4 ± 0.5 (5.3–7.2) | – | – | – | – |
| SE pore | 71.4 ± 2.9 (65.2–76.4) | – | – | – | – |
| Pharynx | 80.2 ± 2.9 (73.7–85.4) | 75 (69–82) | 68 (66–75) | 58–85 | |
| Body width | 12.0 ± 1.0 (10.2–13.5) | – | – | – | – |
| Vulval body diam | 10.6 ± 0.8 (8.9–12.1) | – | – | – | – |
| Anal body diam | 8.3 ± 0.6 (7.3–9.4) | – | – | 8–15 | – |
| Distance from vulva to tail terminus | 60.3 ± 4.6 (51.7–69) | – | – | 79–84 | – |
| Tail length | 31.2 ± 2.3 (26.5–34.8) | – | – | – | – |
aComposite results of six populations.
Notes: All measurements are in µm and in the form of mean ± s.d. (range).
Body slender, not obese, ventrally arcuate when heat relaxed; cuticle finely annulated; lateral field with four incisures; SEM observations showing a somewhat rectangular labial disc with two distinct amphid openings, submedian lobes absent and oral aperture slit like surrounded by semi-globular shaped projections positioned on the labial plate; cephalic region narrow, flattened rounded, not offset from body; cephalic sclerotization weak; stylet short, cone
Not found.
This population was found in the rhizosphere of
The
Figure 3:
Light photomicrographs of
Figure 4:
Scanning electron microscopy of
Measurements of the females of
Morphometric data for
| Characters/ratios | Zhejiang population | Type species |
|---|---|---|
| n | 23 | 10 |
| L | 314.0 ± 17.0 (284.4–352.0) | 260 (230–280) |
| a | 25.0 ± 1.8 (21.6–28.0) | 21 (18–24) |
| b | 4.3 ± 0.2 (3.8–4.7) | 3.8 (3.4–4.3) |
| c | 13.0 ± 2.2 (9.6–17.3) | 12.0 (10–16) |
| c' | 2.9 ± 0.3 (2.3–3.4) | – |
| V | 81.7 ± 0.9 (80.0–83.6) | 81 (73–84) |
| Lip height | 2.8 ± 0.3 (2.2–3.3) | – |
| Lip width | 5.6 ± 0.3 (5.1–6.3) | – |
| Stylet | 23.2 ± 0.9 (21.8–24.8) | 22 (20–24) |
| Stylet percentage | 7.4 ± 0.4 (6.6–7.9) | – |
| Median bulb length | 15.0 ± 1.1 (12.4–16.9) | – |
| Median bulb diam. | 6.6 ± 0.6 (5.7–8.0) | – |
| Ant. end to excretory pore | 66.7 ± 3.1 (61.7–72.5) | – |
| Pharynx | 73.0 ± 2.4 (68.6–77.3) | – |
| Max. body diam. | 12.6 ± 0.9 (10.6–14.3) | – |
| Vulval body diam | 11.0 ± 0.8 (8.4–12.1) | – |
| Anal body diam. | 8.7 ± 1.1 (7.0–11.3) | – |
| Vulva to tail term. | 57.5 ± 4.2 (51.0–65.3) | – |
| Tail length | 25.0 ± 3.9 (18.4–32.5) | – |
Notes: All measurements are in µm and in the form of mean ± s.d. (range).
Body slender, not obese, ventrally arcuate when heat relaxed; cuticle finely annulated; lateral field with four incisures; SEM observations showing a smooth lip region where cuticle elevated at lip margins, submedian lobes absent, oral aperture slit like surrounded by semi-globular shaped projections positioned on the labial plate; cephalic region narrow, truncated, not offset from body; cephalic sclerotization weak; stylet flexible, short, cone
Not found.
This population was found in the rhizosphere of
The
Both species were molecularly characterized using partial 18S, D2-D3 of 28S, and ITS region of rDNA gene and deposited in the GenBank. The genus contains more than 140 species but only a dozen species have been molecularly characterized.
Based on the phylogenetic analysis of the 18S gene (Fig. 5), the pin nematodes were split as two highly supported monophyletic clades with 100% support.
Figure 5:
Tree inferred from 18S under TrN+I+G model (-lnL=4424.0723; AIC=8862.1445; freqA = 0.2337; freqC=0.2276; freqG=0.2917; freqT=0.247;
Based on 28S gene (Fig. 6),
Figure 6:
Bayesian 10001st tree inferred from 28S under GTR+G model (-lnL=5406.0205; AIC=10830.041; freqA=0.2094; freqC=0.2149; freqG=0.3191; freqT=0.2565;
Based on ITS gene (Fig. 7), our population of
Figure 7:
Bayesian 10001st tree inferred from ITS under GTR+I+G model (-lnL=9973.9414; AIC=19967.8828; freqA=0.2117; freqC=0.2617; freqG=0.2691; freqT=0.2574;
Our phylogenetic tree based on 18S, 28S and ITS tree gene sequences revealed that the
Ghaderi et al. (2014) presented a key for the species identification of pin nematodes, in our phylogenetic analysis most of the species belong to the group 2 (stylet < 40 μ m, lateral lines = 3, advulval flaps present); Group 3 (Stylet < 40 μ m, lateral lines = 4, advulval flaps present); Group 7 (stylet > 40 μ m, lateral lines = 2, advulval flaps absent); Group 8 (stylet > 40 μ m, lateral lines = 3, advulval flaps present); Group 9 ( stylet > 40 μ m, lateral lines = 3, advulval flaps absent); Group 10 (Stylet > 40 μ m, lateral lines = 4, advulval flaps present); Group 11( Stylet > 40 μ m; lateral lines = 4, advulval flaps absent).
The host range of pin nematodes in China is quite diverse. The paratylenchid species have been documented from plants, shrubs and trees (Chen et al., 2002; Fang et al., 2012; Wang et al., 2016; Maria et al., 2018a). Out of 31 reported species, only