Updated description of Paratylenchus lepidus Raski 1975 and P. minor Sharma, Sharma and Khan, 1986 by integrating molecular and ultra-structural observations

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Updated description of Paratylenchus lepidus Raski 1975 and P. minor Sharma, Sharma and Khan, 1986 by integrating molecular and ultra-structural observations

Munawar Maria / Wentao Miao / Weimin Ye / Jingwu Zheng *

Keywords : DNA sequencing, morphology, morphometrics, nematode, new record, Pinus species, phylogeny, scanning electron microscopy

Citation Information : Journal of Nematology. Volume 51, Pages 1-13, DOI: https://doi.org/10.21307/jofnem-2019-056

License : (CC-BY-4.0)

Received Date : 28-June-2019 / Published Online: 17-September-2019

ARTICLE

ABSTRACT

Two populations of Paratylenchus lepidus and P. minor were detected in the rhizosphere of Elaeocarpus sp. and Chinese red pine from Taizhou and Hangzhou, Zhejiang Province, China. Previously, P. lepidus has been reported from China whereas P. minor was originally described from India decades ago in the rhizosphere of peach but was never reported thereafter. In this study, both species were characterized morphologically and molecularly coupled with SEM observations. Morphologically, both species have four incisures in the lateral field, vulval present (SEM observations), stylet less than 30 μ m long and cephalic region without submedian lobes. Phylogenetically, both species grouped with paratylenchid species having short stylets. Both species can be differentiated from each other by the shape of lip region (rounded in P. lepidus and narrow truncated in P. minor) and tail terminus (pointed in P. lepidus and a broadly rounded in P. minor) and several morphomemtrical values.The study provided an updated description of P. lepidus and P. minor and a first record of P. minor from China. In addition, both species are the first paratylenchid species recorded from Elaeocarpus sp. and Pinus tabuliformis, respectively.

Graphical ABSTRACT

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. Gracilacus and Paratylenchus based on stylet length (Raski, 1962). The species having longer stylet are cortical feeders while the short stylet species are epidermal feeder (Siddiqi, 2000). The morphological identification of pin nematodes is complicated due to their smaller size and variable characters (Raski, 1962; Brzeski and Hanel, 2000). However several robust characters such as number of lateral lines, stylet length and the presence/absence of vulval flap can sufficiently be used to differentiate pin nematode species (Ghaderi et al., 2014). For precise species identification, rRNA sequences of 28S (Subbotin et al., 2005), 18S (Van Megen et al., 2009), and ITS (Chen et al., 2008, 2009; Van den Berg et al., 2014) have been successfully utilized in pin nematode taxonomy. Recently, several new and known species of nematodes were described based on combined morphological, molecular, and SEM observation (Maria et al., 2018b; Zhuo et al., 2018). Moreover, DNA studies have significant advantages to precisely identify morphologically similar species, such as cryptic species. In an attempt to document the ectoparasitic nematodes from Zhejiang Province, China, two population of Paratylenchus species were detected in a soil sample of Elaeocarpus sp. and Chinese red pine (Pinus tabuliformis Carr). The preliminary studies revealed the status of these species as P. lepidus Raski, 1975a, 1975b and P. minor (Sharma et al., 1986). P. lepidus was originally described from Srilanka and has been reported from China (Chen et al., 2007) whereas P. minor was described from India in the rhizosphere of peach plants and has not been reported thereafter. As these species were reported decades ago, there was no molecular sequencing data and SEM observations. In order to fill these gaps, the objectives of this study were (i) to provide the updated description with morphological and molecular characterization, (ii) to elucidate important morphological details through SEM observations, (iii) to investigate the phylogenetic position and to locate the closely related Paratylenchus species.

Materials and methods

Nematode samplings, extraction, and morphological study

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).

Molecular analyses

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).

Phylogenetic analysis

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.

Results and description

Systematics

Paratylenchus lepidus Raski, 1975a, b

(Figs. 12)

Figure 1:

Light photomicrographs of Paratylenchus lepidus (Raski, 1975a, b). (A) Entire female; (B) lateral lines; (C-D) pharyngeal regions, arrow pointing on the excretory pore (exp); (E) posterior region showing gonad; (F-I) tail regions, arrows pointing on vulva and anus (a) (Scale bars = A = 50 μm, B-I = 10 μm).

10.21307_jofnem-2019-056-f001.jpg
Figure 2:

Scanning electron microscopy of Paratylenchus lepidus (Raski, 1975a, 1975b). (A-C) Lip regions; (D, E) vulval regions; (F, G) posterior region arrows showing position of vulva; (H-K) female tails (Scale bars, A-C = 5 μm; D-K = 10 μm).

10.21307_jofnem-2019-056-f002.jpg

Measurements

Measurements of the females of P. lepidus Taizhou population are given in Table 1.

Table 1.

Morphometric data for Paratylenchus lepidus (Raski, 1975a, 1975b).

10.21307_jofnem-2019-056-t001.jpg

Description

Female

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 ca 70% of the total stylet length; stylet knobs rounded, posteriorly directed; dorsal oesophageal gland opening 5 to 6 μ m behind stylet base; median pharyngeal bulb elongate, bearing distinct large valve; isthmus short slender, surrounded by nerve ring; basal bulb pyriform, cardia inconspicuous; excretory pore position slightly anterior to or at midway of basal pharyngeal bulb; hemizonid position located immediately anterior to the excretory pore; gonad short, prodelphic, spermatheca rounded; vulva a transverse slit occupying half of the body width, vulval lips not protruding with prominent advulval flap; post uterine sac absent; anus indistinct; tail slender, finely annulated, relatively curved, gradually tapers to form a finely rounded to bluntly pointed terminus.

Male and Juveniles

Not found.

Host and locality

This population was found in the rhizosphere of Elaeocarpus sp. from Taizhou City, Zhejiang Province, China. The geographical position of the sampling site is 120°17'23 E; 29°4'30 N.

Remarks

The P. lepidus was originally described from Sri Lanka associated with the tea plants (Raski, 1975b). It has also been reported from Taiwan (Chen et al., 2007) and Hunan (Yu et al., 2014). The Taizhou and Taiwan populations match well with the original description. However, it is presumed that the population from Hunan is not P. lepidus. The large variation in morphometrics and the photo documentation provided by Yu et al. (2014) are not clear enough to support the identification of this population as P. lepidus. Based on the number of lateral lines, stylet length and the presence/absence of vulval flap, Ghaderi et al. (2014) presented a grouping scheme of Paratylenchus species. According to this scheme, P. lepidus belongs to group 3 and have the following specific codes A3, B1, C1, D3, E1, F2.

Paratylenchus minor (Sharma et al., 1986)

(Figs. 34)

Figure 3:

Light photomicrographs of Paratylenchus minor (Sharma et al., 1986). (A) Entire female; (B) lateral lines; (C-D) lip region arrow pointing on the stylet basal knobs (st.k): (F-I) pharyngeal regions, arrow pointing on dorsal pharyngeal gland orifice (dgo), excretory pore (exp), hemizonid (h) and pharyngea basal bulb (ph.b); (J) posterior region showing gonad, arrow pointing on vulva (v); (K) vulval region; (L-O) tail regions, arrows pointing on vulva and anus (a). (Scale bars =A = 50 μm, B-O1 = 0 μm).

10.21307_jofnem-2019-056-f003.jpg
Figure 4:

Scanning electron microscopy of Paratylenchus minor (Sharma et al., 1986). (A) Entire female; (B, C) lip regions; (D) lateral lines; (E) vulval region; (F) posterior region, arrows showing position of vulva (Scale bars, A = 50 μm; B, C, E = 5 μm; D = 10 μm; F = 20 μm).

10.21307_jofnem-2019-056-f004.jpg

Measurements

Measurements of the females of P. minor Hangzhou population are given in Table 2.

Table 2.

Morphometric data for Paratylenchus minor (Sharma et al., 1986).

10.21307_jofnem-2019-056-t002.jpg

Description

Female

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 ca 70% of the total stylet length; stylet knobs rounded; dorsal oesophageal gland opening 5 to 6 μ m behind stylet base; median pharyngeal bulb elongate, bearing distinct large valve; isthmus short slender, surrounded by nerve ring; basal bulb pyriform, cardia inconspicuous; excretory pore position slightly anterior to or at midway of basal pharyngeal bulb; hemizonid position located immediately anterior to the excretory pore; gonad short, prodelphic, spermatheca elongated squarish; vulva a transverse slit occupying half of the body width, vulval lips not protruding with advulval flap; post uterine sac absent; anus indistinct; tail slender, finely annulated, relatively curved, gradually tapers to form a broadly rounded terminus.

Male and Juveniles

Not found.

Host and locality

This population was found in the rhizosphere of Pinus tabuliformis Carr. from Zijingang Campus, Zhejiang University, Hangzhou, Zhejiang Province, China. The geographical position of the sampling site is 120°4'43 E; 30°18'9 N.

Remarks

The P. minor was originally described from India (Sharma et al., 1986) and was not reported thereafter. This population from Hangzhou match well with the original description except for slightly longer body (314 (284-352) vs 260 (230-280) µm) and the presence of advulval flap. The advulval flap has a diagnostic value (Ghaderi et al., 2014), but it is only prominent in some species and difficult to observe under a light microscope. In the Hangzhou population of P. minor, the advulval flap were not discernible under the light microscope, but only visible under the SEM. Based on these characters, the species belongs to the group 3 of Ghaderi et al. (2014) and has the following specific codes A3, B1, C1, D3, E1, F3.

Molecular profiles and phylogenetic status

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. P. lepidus (MK886695) is in a clade with an unidentified Gracilacus (MF095023) species from the USA and P. straelini (AY284631). This clade is sister to a clade comprised of P. conicephalus (KP966493), P. dianthus (AJ966496), P. goldeni (KJ934186), P. microdorus (AY284633), P. nanus (KJ636435), P. similis (KJ636432), P. projectus (KJ636433-34, MF094890), and P. cf. neoamlicephalus (AY284634). All of these species have 4 lateral lines, advulval flap and stylet less than 40 µm except P. straelini has stylet longer than 40 µm. P. minor (MK660189) is in a second clade and is grouped with P. shenzhenensis (KF668497, 3 lateral lines, advulval flap present and stylet less than 40 µm) and it further grouped with species having longer stylet i.e. Gracilacus colinus (KP966494, three lateral lines, advulval flap present), G. paralatescence (MH200615, three lateral lines, advulvul flap absent), and G. wuae (MF095028, four lateral lines, advulval flap absent). Apparently, both species grouped with species having shorter stylets and the presence of advulval flap.

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; R(a)=1; R(b)=2.2828; R(c)=1; R(d)=1; R(e)=7.2373; R(f)=1; Pinva = 0.6811; Shape = 0.7399). Posterior probability values exceeding 50% are given on appropriate clades.

10.21307_jofnem-2019-056-f005.jpg

Based on 28S gene (Fig. 6), P. lepidus (MK886692) is identical with an unidentified Paratylenchus sp. (MH156807) from Fujian Province and it is grouped with unidentified Paratylenchus sp. from Zhejiang (JX109836) and Paratylenchus sp. from USA (KF242230-32). This clade is sister to a clade comprised of P. dianthus (KF242229), P. straeleni (KM875547), P. hamatus (KF242218), P. tenuicaudatus (KU291239), and P. nanus (MH237651, KY468903), all of these species have short stylet, advulval flap and four lateral lines except P. straeleni this species also have advulval flap and four lateral lines but stylet is 44 to 66 μ m long. The pairwise similarity between P. lepidus and unidentified Paratylenchus sp. (MH156807) from Fujian, Paratylenchus sp. (JX109836) from Zhejiang and Paratylenchus sp. from USA (KF242230-32) are 99, 91 and 92% with 2, 59 and 64 bp difference.

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; R(a)=0.5351; R(b)=2.123; R(c)=0.9422; R(d)=0.4173; R(e)=4.3792; R(f)=1; Pinva = 0; Shape = 0.3021). Posterior probability values exceeding 50% are given on appropriate clades.

10.21307_jofnem-2019-056-f006.jpg

P. minor (MK660188) is nearly identical with an unidentified Paratylenchus species (KT258978) from Guangdong Province. This clade is grouped with P. aquaticus (KF2422439-41), P. leptos (KR270602) and P. rostrocaudatus (KR270601). These species have short stylet and have advulval flap, but the number of lateral lines is not the same, i.e. 3 for P. leptos and P. rostrocaudatus, and 2 for P. aquaticus. This clade is further grouped with species having longer stylets and absence of advulval flaps but the number of lateral lines is not consistent, i.e. 3 for P. aculentus and G. paralatescence and 4 for the G. wuae. The pairwise similarity between P. minor and unidentified Paratylenchus sp. (KT258978) from Guangdong is 99% with 9 bp difference.

Based on ITS gene (Fig. 7), our population of P. lepidus (MK886695) is identical with P. lepidus (EF126178) from Taiwan and further grouped with P. lepidus (JX992859-60) from Hunan and an unidentified Paratylenchus sp. (KF242273-74) from USA, but with significant difference based on branch length. P. minutus (EF126180) is in the same clade as P. lepidus from Hunan. P. dianthus (LC462227 and LC462228, KF242272) is sister to this clade. This clade is further grouped with the unidentified Paratylenchus (KF242243) species from the USA, P. hamatus (KF242257), P. nanus (MH236098, KY4668906), P. labiosus (JQ708154) and P. goldeni (KJ934186). All of these species are reported to have shorter stylet, advulal present and four lateral lines. The ITS tree revealed that the P. lepidus from Hunan might be a different species, as indicated by the ITS pairwise sequence identity between P. lepidus from Zhejiang and Hunan population, i.e. only 83% identical with 156 bp difference. Considering this we propose that P. lepidus was only reported from Taiwan and Zhejiang. The two unidentified Paratylenchus species (KF242273-74) from the USA possibly be similar to that of P. lepidus from Hunan. The pairwise sequence similarities of P. lepidus from Hunan and Paratylenchus species (KF242273-74) from USA is 99% with 2 bp difference.

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; R(a)=1.3546; R(b)=2.6646; R(c)=1.6199; R(d)=0.7276; R(e)=3.229; R(f)=1; Pinva=0.3363; Shape=1.6939). Posterior probability values exceeding 50% are given on appropriate clades.

10.21307_jofnem-2019-056-f007.jpg

P. minor is nearly identical with an unidentified Paratylenchus species (KT258979) from Guangdong Province and it is sister to P. leptos (KR270605) and P. rostrocaudatus (KR270604), both of these species have short stylets and advulval flap but the number of lateral lines is three. This clade is further grouped with the species having longer stylets, i.e. G. paralatecence (MH200615), P. aculentus (KR270603, EU247526), P. nanjingensis (KM 366103), G. wuae (KM061783), P. guangzhouensis (KT725653), G. bilineata (EU247525), and P. aquaticus (KF242278). The pairwise sequence identities between P. minor and unidentified Paratylenchus species (KT258979) from Guangdong Province is 98% with 10 bp difference.

Our phylogenetic tree based on 18S, 28S and ITS tree gene sequences revealed that the P. lepidus and P. minor are in two well-separated clades and grouped with species having shorter stylets and advuval flap, the species having longer stylets arranged at the basal position of the trees. To this point we suppose only fewer paratylenchid sequences were available for the phylogenetic studies, the inclusion of more sequences from other known species will be helpful to provide a better resolution of paratylenchid phylogeny.

Additional remarks

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 P. alleni (Raski, 1975a); P. bukowinensis (Micoletzky, 1922); P. epacris ( Allen and Jensen, 1950); P. minutus (Linford et al., 1949); P. nanjingensis (Wang et al., 2014), and G. steineri (Golden, 1961) were documented from Pinus massoniana, P. sylvestris and P. thunbergii (Chen et al., 2002; Fang et al., 2012). In this regard, P. minor is the first paratylenchid species found in the rhizosphere of Pinus tabuliformis which is a native pine species of northern and southern China (Farjon, 2013). None of the Paratylenchus species was ever found from the Elaeocarpus sp. in China. The discovery of these two pin nematodes expanded the distribution and host range. It is also noted that the recent discovery of several new species of paratylenchids from China suggests that presumably only a small fraction of the actually existing taxa is known so far. There are still large geographical areas insufficiently studied for their diversity.

Acknowledgments

This research was supported by the National Natural Science Foundation of China (Project No. 31772137). The authors thank Dr N. H. Rong from the Center of Electron Microscopy, Life Science Division, Zhejiang University for providing assistance in the preparation of SEM.

References


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  20. Olson, M. , Harris, T. , Higgins, R. , Mullin, P. , Powers, K. , Olson, S. and Powers, T. O. 2017. Species delimitation and description of Mesocriconema nebraskense n. sp (Nematoda: Criconematidae), a morphologically cryptic, parthenogenetic species from North American Grasslands. Journal of Nematology 49:42–66.
  21. Powers, T. O. , Harris, T. , Higgins, R. , Sutton, L. and Powers, K. S. 2010. Morphological and molecular characterization of Discocriconemella inarata, an endemic nematode from North American native tallgrass prairies. Journal of Nematology 42:35–45.
  22. Raski, D. J. 1962. Paratylenchidae n. fam. with descriptions of five new species of Gracilacus n. g. and an emendation of Cacopaurus Thorne, 1943, Paratylenchus Micoletzky, 1922 and Criconematidae Thorne, 1943. Proceedings of the Helminthological Society of Washington 29:189–207.
  23. Raski, D. J. 1975a. Revision of the genus Paratylenchus Micoletzky, 1922 and descriptions of new species. Part I of three parts. Journal of Nematology 7:15–34.
  24. Raski, D. J. 1975b. Revision of the genus Paratylenchus Micoletzky, 1922 and descriptions of new species. Part II of three parts. Journal of Nematology 3:274–95.
  25. Raski, D.J. 1991. Tylenchulidae in agricultural soils. in Nickle, W.R. (Eds), Manual of agricultural nematology. Marcel Dekker, New York, NY: 761–94.
  26. Seinhorst, J. W. 1959. A rapid method for the transfer of nematodes from fixative to anhydrous glycerin. Nematologica 4:67–69.
  27. Sharma, G. C. , Sharma, N. K. and Khan, E. 1986. Two new species of genus Paratylenchus Micoletzky, 1922 (Nematoda: Paratylenchinae) from Himachal Pradesh, India. Indian Journal of Nematology 16:231–35.
  28. Siddiqi, M.R. 2000. Tylenchida: parasites of plants and insects. 2nd ed., CABI Publishing, Wallingford:: 833pp.
  29. Subbotin, S. A. , Madani, M. , Krall, E. , Sturhan, D. and Moens, M. 2005. Molecular diagnostics, taxonomy, and phylogeny of the stem nematode Ditylenchus dipsaci species complex based on the sequences of the internal transcribed spacer-rDNA. Phytopathology 95:1308–15.
  30. Van den Berg, E. , Tiedt, L. R. and Subbotin, S. A. 2014. Morphological and molecular characterisation of several Paratylenchus Micoletzky, 1922 (Tylenchida: Paratylenchidae) species from South Africa and USA, together with some taxonomic notes. Nematology 16:323–58.
  31. Van Megen, H. , van den Elsen, S. , Holterman, M. , Karssen, G. , Mooyman, P. , Bongers, T. , Holovachov, O. , Bakker, L. and Helder, J. 2009. A phylogenetic tree of nematodes based on about 1200 full-length small subunit ribosomal DNA sequences. Nematology 11:927–50.
  32. Wang, K. , Li, Y. , Xie, H. , Xu, C. L. and Wu, W. J. 2016. Morphology and molecular analysis of Paratylenchus guangzhouensis n. sp. (Nematoda: Paratylenchinae) from the soil associated with Bambusa multiplex in China. European Journal of Plant Pathology. Journal of Nematology 48:255–64.
  33. Wang, K. , Xie, H. , Li, Y. , Wu, W. J. and Xu, C. L. 2014. Morphology and molecular analysis of Paratylenchus nanjingensis n. sp. (Nematoda: Paratylenchinae) from the rhizosphere soil of Pinus massoniana in China. Journal of Helminthology 90:166–73.
  34. Ye, W. , Giblin-Davis, R. M. , Braasch, H. , Morris, K. and Thomas, W. K. 2007. Phylogenetic relationships among Bursaphelenchus species (Nematoda: Parasitaphelenchidae) inferred from nuclear ribosomal and mitochondrial DNA sequence data. Molecular Phylogenetics and Evolution 43:1185–97.
  35. Yokoo, T. 1970. Studies on the nemic-fauna of soil of the mulberry plant field in Kyushu. II. On the occurrence of two pin nematodes, Paratylenchus aciculus Brown, 1959 and P. morius n. sp. Agricultural Bulletin of Saga University 30:1–10.
  36. Yu, Y. , Liu, H. , Zeng, L. , Zhang, G. and Zhu, A. 2014. New record of paratylenchus lepidus (nematoda: tylenchulidae) associated with ramie root inYuanjiang, Hunan Province, China. Pakistan journal of Zoology 46:583–386.
  37. Zheng, J. , Subbotin, S. A. , He, S. , Gu, J. and Moens, M. 2003. Molecular characterization of some Asian isolates of Bursaphelenchus xylophilus and B. mucronatus using PCR-RFLPs and sequences of ribosomal DNA. Russian Journal of Nematology 11:17–22.
  38. Zhuo, K. , Liu, X. , Tao, Ye. , Wang, H. , Lin, B. and Liao, J. 2018. Morphological and molecular characterisation of three species of Paratylenchus Micoletzky, 1922 (Tylenchida: Paratylenchidae) from China, with a first description of the male P. rostrocaudatus. Nematology 20:837–50.
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FIGURES & TABLES

Figure 1:

Light photomicrographs of Paratylenchus lepidus (Raski, 1975a, b). (A) Entire female; (B) lateral lines; (C-D) pharyngeal regions, arrow pointing on the excretory pore (exp); (E) posterior region showing gonad; (F-I) tail regions, arrows pointing on vulva and anus (a) (Scale bars = A = 50 μm, B-I = 10 μm).

Full Size   |   Slide (.pptx)

Figure 2:

Scanning electron microscopy of Paratylenchus lepidus (Raski, 1975a, 1975b). (A-C) Lip regions; (D, E) vulval regions; (F, G) posterior region arrows showing position of vulva; (H-K) female tails (Scale bars, A-C = 5 μm; D-K = 10 μm).

Full Size   |   Slide (.pptx)

Figure 3:

Light photomicrographs of Paratylenchus minor (Sharma et al., 1986). (A) Entire female; (B) lateral lines; (C-D) lip region arrow pointing on the stylet basal knobs (st.k): (F-I) pharyngeal regions, arrow pointing on dorsal pharyngeal gland orifice (dgo), excretory pore (exp), hemizonid (h) and pharyngea basal bulb (ph.b); (J) posterior region showing gonad, arrow pointing on vulva (v); (K) vulval region; (L-O) tail regions, arrows pointing on vulva and anus (a). (Scale bars =A = 50 μm, B-O1 = 0 μm).

Full Size   |   Slide (.pptx)

Figure 4:

Scanning electron microscopy of Paratylenchus minor (Sharma et al., 1986). (A) Entire female; (B, C) lip regions; (D) lateral lines; (E) vulval region; (F) posterior region, arrows showing position of vulva (Scale bars, A = 50 μm; B, C, E = 5 μm; D = 10 μm; F = 20 μm).

Full Size   |   Slide (.pptx)

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; R(a)=1; R(b)=2.2828; R(c)=1; R(d)=1; R(e)=7.2373; R(f)=1; Pinva = 0.6811; Shape = 0.7399). Posterior probability values exceeding 50% are given on appropriate clades.

Full Size   |   Slide (.pptx)

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; R(a)=0.5351; R(b)=2.123; R(c)=0.9422; R(d)=0.4173; R(e)=4.3792; R(f)=1; Pinva = 0; Shape = 0.3021). Posterior probability values exceeding 50% are given on appropriate clades.

Full Size   |   Slide (.pptx)

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; R(a)=1.3546; R(b)=2.6646; R(c)=1.6199; R(d)=0.7276; R(e)=3.229; R(f)=1; Pinva=0.3363; Shape=1.6939). Posterior probability values exceeding 50% are given on appropriate clades.

Full Size   |   Slide (.pptx)

REFERENCES

  1. Allen, M.W. and Jensen, H.J. 1950. Cacopaurus epacris, new species (Nematoda: Criconematidae), a nematode parasite of California black walnut roots. Proceedings of the Helminthological Society of Washington 17:10–14.
  2. Brzeski, M. W. and Hanel, L. 2000. Paratylenchinae: evaluation of diagnostic morpho-biometrical characters of females in the genus Paratylenchus Micoletzky, 1922 (Nematoda: Tylenchulidae). Nematology 2:253–61.
  3. Chen, D. Y. , Ni, H. F. , Tsay, T. T. and Yen, J. H. 2008. Identification of Gracilacus bilineata and G. aculenta (Nematoda: Criconematoidea, Tylenchulidae) among bamboo plantations in Taiwan. Plant Pathology Bulletin 17:209–19, (In Chinese).
  4. Chen, D. Y. , Ni, H. F. , Yen, J. H. and Tsay, T. T. 2007. Identification of a new recorded pin nematode, Paratylenchus lepidus, (Nematoda: Criconematoidea, Tylenchulidae) among tea plantations in Taiwan. Plant Pathology Bulletin 16:41–46, (In Chinese).
  5. Chen, D. Y. , Ni, H. F. , Yen, J. H. and Tsay, T. T. 2009. Identification of a new recorded pin nematode Paratylenchus minutus (Nematoda: Criconematoidea, Tylenchulidae) in Taiwan. Plant Pathology Bulletin 18:167–74, (In Chinese).
  6. Chen, L. , Liu, W. and Qin, B. 2002. Host plants and distribution of Paratylenchus (Tylenchida : Criconematoidea) in China. Liaoning Agriculture Sciences 3:4–8, In Chinese).
  7. De Ley, P. , Félix, M.A. , Frisse, L. M. , Nadler, S. A. , Sternberg, P. W. and Thomas, W. K. 1999. Molecular and morphological characterization of two reproductively isolated species with mirror-image anatomy (Nematoda: Cephalobidae). Nematology 2:591–612.
  8. Fang, A. , Tan, J. , Ye, J and Hao, D. 2012. Species identification of rhizosphere parasitic nematodes of pine tree in Jiangsu Province. Journal of Nanjing Forestry University 36:109–14, (In Chinese).
  9. Farjon, A. 2013. Pinus tabuliformis. The IUCN Red List of Threatened Species. IUCN. 2013. e.T42419A2978916. doi:10.2305/IUCN.UK.2013-1.RLTS.T42419A2978916.en.
  10. Ghaderi, R. , Kashi, L. and Karegar, A. 2014. Contribution to the study of the genus Paratylenchus Micoletzky, 1922 sensu lato (Nematoda: Tylenchulidae). Zootaxa 3814:151–87.
  11. Golden, A. M. 1961. Paratylenchus steineri (Criconematidae) a new species of plant nematode. Proceedings of the Helminthological Society of Washington 28:9–11.
  12. Jenkins, W. R. 1964. A rapid centrifugal-flotation technique for separating nematodes from soil. Plant Disease Reporter 48:692.
  13. Joyce, S. , Reid, A. , Driver, F. and Curran, J. 1994. Application of polymerase chain reaction (PCR) methods to identification of entomopathogenic nematodes. in Burnell, A. M. , Ehlers, R. U. and Masson, J. P (Eds), COST 812 Biotechnology: Genetics of Entomopathogenic Nematode-bacterium Complexes. Proceedings of Symposium & Workshop, St. Patrick’s College, Maynooth, Co, European Commission, DG XII, Kildare, Ireland, Luxembourg: 178–87.
  14. Khan, E. , Prasad, S. K. and Mathur, V. K. 1967. Two new species of the genus Paratylenchus Micoletzky, 1922 (Nematoda: Criconematidae) from India. Nematologica 13:79–84.
  15. Linford, M. B. , Oliveira, J. M. and Ishii, M. 1949. Paratylenchus minutus n. sp., a nematode parasitic on roots. Pacific Science 3:111–19.
  16. Maria, M. , Cai, R. , Castillo, P. and Zheng, J. 2018c. Morphological and molecular characterization of Hemicriconemoides paracamelliae sp. n. (Nematoda: Criconematidae) and two known species of Hemicriconemoides from China. Nematology 20:403–22.
  17. Maria, M. , Powers, T. O. , Tian, Z. and Zheng, J. 2018b. Distribution and description of criconematids from Hangzhou, Zhejiang Province, China. Journal of Nematology 50:183–206.
  18. Maria, M. , Ruihang, C. , Ye, W. , Powers, T. O. and Zheng, J. 2018a. Description of Gracilacus paralatescens n. sp. (Nematoda: Paratylenchinae) found from the rhizosphere of bamboo in Zhejiang, China. Journal of Nematology 50:611–22.
  19. Micoletzky, H. 1922. Die Freilebenden Erd-Nematoden. Archiv für Naturgeschichte, Berlin A. 87:1–650.
  20. Olson, M. , Harris, T. , Higgins, R. , Mullin, P. , Powers, K. , Olson, S. and Powers, T. O. 2017. Species delimitation and description of Mesocriconema nebraskense n. sp (Nematoda: Criconematidae), a morphologically cryptic, parthenogenetic species from North American Grasslands. Journal of Nematology 49:42–66.
  21. Powers, T. O. , Harris, T. , Higgins, R. , Sutton, L. and Powers, K. S. 2010. Morphological and molecular characterization of Discocriconemella inarata, an endemic nematode from North American native tallgrass prairies. Journal of Nematology 42:35–45.
  22. Raski, D. J. 1962. Paratylenchidae n. fam. with descriptions of five new species of Gracilacus n. g. and an emendation of Cacopaurus Thorne, 1943, Paratylenchus Micoletzky, 1922 and Criconematidae Thorne, 1943. Proceedings of the Helminthological Society of Washington 29:189–207.
  23. Raski, D. J. 1975a. Revision of the genus Paratylenchus Micoletzky, 1922 and descriptions of new species. Part I of three parts. Journal of Nematology 7:15–34.
  24. Raski, D. J. 1975b. Revision of the genus Paratylenchus Micoletzky, 1922 and descriptions of new species. Part II of three parts. Journal of Nematology 3:274–95.
  25. Raski, D.J. 1991. Tylenchulidae in agricultural soils. in Nickle, W.R. (Eds), Manual of agricultural nematology. Marcel Dekker, New York, NY: 761–94.
  26. Seinhorst, J. W. 1959. A rapid method for the transfer of nematodes from fixative to anhydrous glycerin. Nematologica 4:67–69.
  27. Sharma, G. C. , Sharma, N. K. and Khan, E. 1986. Two new species of genus Paratylenchus Micoletzky, 1922 (Nematoda: Paratylenchinae) from Himachal Pradesh, India. Indian Journal of Nematology 16:231–35.
  28. Siddiqi, M.R. 2000. Tylenchida: parasites of plants and insects. 2nd ed., CABI Publishing, Wallingford:: 833pp.
  29. Subbotin, S. A. , Madani, M. , Krall, E. , Sturhan, D. and Moens, M. 2005. Molecular diagnostics, taxonomy, and phylogeny of the stem nematode Ditylenchus dipsaci species complex based on the sequences of the internal transcribed spacer-rDNA. Phytopathology 95:1308–15.
  30. Van den Berg, E. , Tiedt, L. R. and Subbotin, S. A. 2014. Morphological and molecular characterisation of several Paratylenchus Micoletzky, 1922 (Tylenchida: Paratylenchidae) species from South Africa and USA, together with some taxonomic notes. Nematology 16:323–58.
  31. Van Megen, H. , van den Elsen, S. , Holterman, M. , Karssen, G. , Mooyman, P. , Bongers, T. , Holovachov, O. , Bakker, L. and Helder, J. 2009. A phylogenetic tree of nematodes based on about 1200 full-length small subunit ribosomal DNA sequences. Nematology 11:927–50.
  32. Wang, K. , Li, Y. , Xie, H. , Xu, C. L. and Wu, W. J. 2016. Morphology and molecular analysis of Paratylenchus guangzhouensis n. sp. (Nematoda: Paratylenchinae) from the soil associated with Bambusa multiplex in China. European Journal of Plant Pathology. Journal of Nematology 48:255–64.
  33. Wang, K. , Xie, H. , Li, Y. , Wu, W. J. and Xu, C. L. 2014. Morphology and molecular analysis of Paratylenchus nanjingensis n. sp. (Nematoda: Paratylenchinae) from the rhizosphere soil of Pinus massoniana in China. Journal of Helminthology 90:166–73.
  34. Ye, W. , Giblin-Davis, R. M. , Braasch, H. , Morris, K. and Thomas, W. K. 2007. Phylogenetic relationships among Bursaphelenchus species (Nematoda: Parasitaphelenchidae) inferred from nuclear ribosomal and mitochondrial DNA sequence data. Molecular Phylogenetics and Evolution 43:1185–97.
  35. Yokoo, T. 1970. Studies on the nemic-fauna of soil of the mulberry plant field in Kyushu. II. On the occurrence of two pin nematodes, Paratylenchus aciculus Brown, 1959 and P. morius n. sp. Agricultural Bulletin of Saga University 30:1–10.
  36. Yu, Y. , Liu, H. , Zeng, L. , Zhang, G. and Zhu, A. 2014. New record of paratylenchus lepidus (nematoda: tylenchulidae) associated with ramie root inYuanjiang, Hunan Province, China. Pakistan journal of Zoology 46:583–386.
  37. Zheng, J. , Subbotin, S. A. , He, S. , Gu, J. and Moens, M. 2003. Molecular characterization of some Asian isolates of Bursaphelenchus xylophilus and B. mucronatus using PCR-RFLPs and sequences of ribosomal DNA. Russian Journal of Nematology 11:17–22.
  38. Zhuo, K. , Liu, X. , Tao, Ye. , Wang, H. , Lin, B. and Liao, J. 2018. Morphological and molecular characterisation of three species of Paratylenchus Micoletzky, 1922 (Tylenchida: Paratylenchidae) from China, with a first description of the male P. rostrocaudatus. Nematology 20:837–50.

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