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

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).

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).

DNA was extracted from several specimens using the proteinase K protocol. DNA extraction, PCR, and cloning protocols were as described by Tanha Maafi et al. (2003). The primer sets: D2A (5′ – ACA AGT ACC GTG AGG GAA AGT TG – 3′) and D3B (5′ – TCG GAA GGA ACC AGC TAC TA – 3′) amplifying the D2-D3 of 28S rRNA gene (Subbotin et al., 2006), TW81 (5′ – GTT TCC GTA GGT GAA CCT GC – 3′) and AB28 (5′ – ATA TGC TTA AGT TCA GCG GGT – 3′) amplifying ITS rRNA (Tanha Maafi et al., 2003) were used in this study. PCR products were purified using the QIAquick Gel Extraction Kit (Qiagen) following the manufacturer’s instructions and submitted to direct sequencing at GENEWIZ (USA, CA). The new Paratylenchus sequences were submitted to the GenBank database under accession numbers: MW413581, MW413582 (28S rRNA gene), and MW413587 (ITS rRNA gene).

The new sequences for each gene (D2–D3 of 28S rRNA, ITS rRNA) were aligned using ClustalX 1.83 (Thompson et al., 1997) with previous published DNA sequences (Etongwe et al., 2020; Munawar et al., 2018; Mwamula et al., 2020; Singh et al., 2021; Subbotin et al., 2005, 2006; Van den Berg et al., 2014; Wang et al., 2016a, b). ClustalX with the modified parameters (gap opening = 5 and gap extension = 3) were applied to generate the D2–D3 of 28S rRNA and ITS rRNA gene alignments. Sequence datasets were analyzed with Bayesian inference (BI) (Ronquist and Huelsenbeck, 2003) using MrBayes 3.1.2 as described by Van den Berg et al. (2014).

Associated with roots and soil of Virginia pine (Pinus virginiana Mill) trees in the Little Paint Branch Park, Beltsville, Prince George’s County, Maryland, USA. The global positioning coordinates: 39.036071 N, 76.391826 W.

Holotype (1 female): Slide T-743t and T-744t (one male), 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-7463p–T-7470p. Additional females on slide numbers T-7471-p–T-7472p at Plant Pest Diagnostic Center, California Department of Food and Agriculture, Sacramento, CA, USA; T-7473p to T-7474 pat University of California, Riverside, CA, USA; T-7475p to T-7476p, University of California, Davis, CA, USA and T-7477p-T-7478p at Fera, Plant Pest Disease Cultures and Collections, York, United Kingdom.

Paratylenchus beltsvillensis n. sp. is characterized by a combination of the following morphological features in females: slender vermiform body (0.24–0.67) mm, long stylet (70–75 µm) with anchor shaped knobs, excretory pore located at the metacorpus level or slightly posterior to it (at 90–100 µm from anterior end), and vulva located at 70–73% with small vulval flap; tail conical, tapering uniformly to a bluntly rounded tail terminus; males have stylet and pharynx degenerate, spicules measuring 17–20 µm and gubernaculum 5.0–5.5 µm.

Paratylenchus beltsvillensis n. sp. is similar with P. nanjingensis and P. aculentus, from which it differs by stylet length (70.0–75.0 vs 64–68 and 48–70 µm) and position of excretory pore (90.0–100.0 vs 55.5–64.5 and 59–89 µm).

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).

It differs from P. aciculus (Brown, 1959) by having a slightly longer stylet (70–75 vs 61–69 µm), a slightly shorter body length (245–267 vs 240–310 µm), and by the males having a longer spicules (17–20 vs 14.5–16.5 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.

It also comes close to P. steneri Golden, 1961 from which it differs by the number of lateral lines (2–3 vs. 4), longer stylet length (70–75 vs. 65–69 µm), and males present vs absent in P. steineri.

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