Morphological and molecular characterization of Paratylenchus beltsvillensis n. sp. (Tylenchida: Paratylenchidae) from the rhizosphere of pine tree (Pinus virginiana Mill) in Maryland, USA

Abstract The pin nematode, Paratylechus beltsvillensis n. sp. collected from rhizosphere soil of a Virginia pine tree (Pinus virginiana Mill) growing in Little Paint Branch Park, Beltsville, Prince George’s County, Maryland, USA, is described and illustrated along with light and scanning electron photomicrographs. Females, males, and juveniles of this new species were recovered from soil samples using the sugar centrifugal flotation and Baermann funnel extraction methods. Morphologically, females are short, body length ranging from 245 to 267 μm, stylet from 70 to 75 μm long with anchor shaped knobs, vulva located at 70–73% and small vulval flap, spermatheca large, and ovoid filled with sperms. Lateral field with three incisures, of which the outer two are prominent. Tail slender, having a rounded tail terminus. Males without stylet and have a degenerated pharynx, spicules = 17–20 µm and gubernaculum = 5.0–5.5 µm. Both morphological observations and molecular analysis of ITS and partial 28S ribosomal RNA gene sequences indicated that the specimens collected from the soil at Beltsville Park from rhizosphere soil samples from Virginia pine represents a new pin nematode species.

The genus Paratylenchus Micoletzky, 1922 is comprised of nematode that are obligate ectoparasites of plants, widely distributed world-wide and associated with a large variety of plants (Ghaderi et al., 2016;Raski, 1991;Siddiqi, 2000;Van den Berg et al., 2014). The first comprehensive review of the genus was given by Tarjan (1960), who made an improvement of the genus diagnosis as well as synonymization of some species. The genus Gracilacus  was proposed for Paratylenchus species with stylet lengths longer than 48 µm, the excretory pore anterior to nerve ring, and well-developed stylet in juveniles . However, Brzeski (1963) suggested a synonymization of Gracilacus with Paratylenchus because the pro posed diagnostic characters were unreliable for defining the genera. Although some authors concurred with the synonymy (Brzeski, 1998;Ghaderi et al., 2016;Siddiqi and Goodey, 1964), others accepted Gracilacus as a valid genus (Abdel-Rahman and Maggenti, 1988;Brzeski, 1995;Doucet, 1994;Esser, 1992;Geraert, 1965;Raski, 1991;Shahina and Maqbool, 1993;Van den Berg and Buckley, 1993) or a subgenus of Paratylenchus (Siddiqi, 2000). In the book on Tylenchulidae, Ghaderi et al. (2016) recognized 117 species of Paratylenchus.
With the advent of molecular biology, phylogenetic studies have been conducted to examine the relationships among paratylenchids. Subbotin et al. (2005) were the first to provide molecular characterization of several Paratylenchus species using partial 28S rRNA gene sequences. Lopez et al. (2013) used the ITS1 rRNA gene to reconstruct paratylenchid relationships including G. bilineata Brzeski (1995) and G. aculenta (Brown, 1959;. Van Den Berg et al. (2014) inferred phylogenetic relationships among several Paratylenchus spp. using 28S rRNA (58 sequences) and ITS rRNA (40 sequences) gene sequences for this genus. Wang et al. (2016a), also using the ITS rRNA gene, demonstrated that their newly described species P. nanjingensis which has a 64-68 µm long stylet grouped with P. bilineatus and P. aculentus. In another study, Wang et al. (2016b) described P. guangzhouensis, a species with the stylet averaging 47 µm long and based on their ITS rRNA phylogeny, the authors showed that this species was clustered with those four species having a long stylet. Recently, Munawar et al. (2021), Singh et al. (2021), Clavero-Camacho et al. (2021) published comprehensive phylogenies of the genus Paratylenchus. These phylo genetic analyses did not support a justification of erection for the genus Gracilacus and this genus was considered as a synonym of Paratylenchus.
During a nematological survey, an unknown Paratylenchus species with a long stylet was found in a rhizosphere soil of a Virginia pine tree (Pinus virginiana Mill) in Beltsville, Prince George's County, Maryland, USA. Morphological and molecular examination of nematode specimens revealed that they belong to a new species, which named here Paratylechus beltsvillensis n. sp. The objective of this study was also to describe this new species using light (LM) and scanning electron microscopy (SEM) and provide its molecular characterization.

Nematode samples
In September and October of 2020, few soil samples were collected in the Little Paint Branch Park, Beltsville, Prince George's County, Maryland, USA And sent to the Plant Pest Diagnostic Center, California Department of Food and Agriculture, Sacramento, CA and part of the same soil samples were analyzed at the Mycology and Nematology Genetic Diversity and Biology Laboratory USDA, ARS (MNGDBL), Beltsville. Nematodes were recovered from soil samples using the sugar centrifugal flotation and Baermann Funnel extraction methods (Jenkins, 1968).

Morphological examination
Nematodes were fixed in 3% formaldehyde and processed to glycerin by the formalin glycerin method (Golden, 1990;Hooper, 1970). Photomicrographs of the specimens were taken with a Nikon Eclipse Ni compound microscope using a Nikon DS-Ri2 camera. Specimens were measured with an ocular micrometer on Leitz DMRB compound microscope. Nematodes were observed with the low-temperature scanning electron microscopy (LT-SEM) using the techniques described in Kantor et al. (2020) and Carta et al. (2020).

Type material
Holotype (1 female
The new species is similar to P. peperpotti (Schoemaker, 1963), although it differs from the latter by having a slightly smaller b value (1.99-2.2 vs 2.0-3.9) and a slightly posterior location of excretory pore (90.0-100.0 vs 61-87 µm).
Also, P. beltsvillensis n. sp. it is close to P. solvaga (Raski, 1976) and P. marylandicus Jenkins, 1960. From P. solvaga, it differs by its tail shape, which is mostly deformed, and location of excretory pore. From P. marylandicus it differs mostly by shorter body length, number of lateral lines (2-3 vs 4), tail shape.

Etymology
The species name is derived from Beltsville, the type locality of this species.

Molecular analysis
The D2-D3 of 28S rRNA gene The alignment generated with modified parameters was 782 bp in length and contained 36 sequences, including two sequences of new species and three sequences of outgroup taxa. Phylogenetic relationships of P. beltsvillensis n. sp. within selected Paratylenchus are given in Figure 4. Sequences of P. beltsvillensis n. sp. formed a clade with that of P. nanjingensis. (KR232932) collected from soil associated with Pinus massoniana in Nanjing, China (Maria et al., unpublished) and differed from this sequence in 27 bp (3.9%).

The ITS rRNA gene
The alignment generated with modified parameters was 1,021 bp in length and contained 21 sequences, including for two outgroups. Phylogenetic relationships of P. beltsvillensis n. sp. within selected Paratylenchus are given in Figure 5. The sequence of P. beltsvillensis n. sp. was inferred to form a clade with that of P. nanjingensis and was different from sequences of P. nanjingensis, P. paralatescens, and P. aculentus type A and P. aculentus type B in 47 bp (6.8%), 51 bp (7.4%) 60 bp (8.7%), and 50 bp (7.2%), respectively.
The phylogenetic relationships within Paratylenchus species obtained in this study are congruent with those recently presented by Zhuo et al. (2018), Munawar et al. (2021), Singh et al. (2021) and Clavero-Camacho et al. (2021). Pratylenchus beltsvillensis n. sp. clustered with species belonging to the Clade II according to Singh et al. (2021). Paratylenchus beltsvillensis n. sp. is molecularly close to P. nanjingensis, P. aculentus, Figure 4: Phylogenetic relationships of Paratylenchus beltsvillensis n. sp. with other related species. Bayesian 50% majority rule consensus tree from two runs as inferred from analysis of the D2-D3 of 28S rRNA gene sequence alignment under the GTR + I + G model. Posterior probabilities equal or more than 70% are given for appropriate clades. New sequences are indicated in bold. Clade numbers are given according to Singh et al. (2021). *identified as Paratylenchus sp. in the GenBank.
In conclusion, both morphological and molecular observations with known and closely related species indicate that Paratylenchus species isolated from rhizosphere soil of a Virginia pine tree represents Figure 5: Phylogenetic relationships of Paratylenchus beltsvillensis n. sp. with other related species: Bayesian 50% majority rule consensus tree from two runs as inferred from analysis of the ITS rRNA gene sequence alignment under the GTR + I + G model. Posterior probabilities equal or more than 70% are given for appropriate clades. New sequences are indicated in bold. Clade numbers are given according to Singh et al. (2021).
a new pin nematode species, described here as Paratylenchus beltsvillensis n. sp.