A nematode species associated with wood samples from dead pine trees (Pinus armandii Franch.) was collected from Shenyang, Liaoning Province, China, during July 2018. It was preliminarily observed by light microscope that the nematode population in the dead pine was sizeable and singular. The nematode was described and illustrated based on its morphological and molecular characteristics in order to identify the species and study its relationship with pine wilt disease. The continuous development of molecular biology allows us to supplement the database of Laimaphelenchus species, leading to the improved disease diagnosis and taxonomy, and providing a reference for pine wilt research.

Wood samples were sliced into approximately 1 cm × 1 cm slices, and nematodes were collected using the Baermann funnel method at room temperature for 8-12 h. Nematodes to be prepared for permanent slides were killed by immersion in 4% formaldehyde solution and immediately fixed. A certain number of nematodes were rinsed with sterile water three times and then transferred into a potato dextrose agar medium containing Botrytis cinerea. After being placed in the incubator for dark culture at 25℃ for 7 days, a large number of nematodes were obtained. Measurements, descriptions and light micrographs of nematodes were carried out and De Man’s formula was used to measure morphological traits and the line was drawn based on these characteristics. Scanning electron microscopy was used to observe the nuances of the lateral field, lip region, vulva and mucronatus. Nematode DNA was extracted and its 18S and 28S D2-D3 regions were amplified, cloned and sequenced. The phylogenetic tree construction used Neighbor-Joining (NJ) methods and an bootstrap analysis was performed to test the support of each node in the tree. The nematode was identified by systematic morphological observation, morphometric values and the comparative analysis of molecular biological sequences.

The nematode was cultured successfully on B. cinerea in the laboratory, indicating that it can feed on fungi. Females are characterized by a 1 252-1 722 μm body length, a slender stylet of 12-16 μm, four incisures in the lateral field, a vulva with a flap, and a tail-tip structure comprising a single stalk-like terminus and two tubercles with four to six finger-like protrusions. Males have spicules 24-30 μm in length, two pairs of caudal papillae, and a tail morphologically similar to that of the female but more ventrally curved. Phylogenetic analyses were based on the D2-D3 expansion segments of the 28S and 18S rRNA gene. Construction of the phylogenetic tree used multi-gene tandem sequences of Laimaphelenchus liaoningensis n. sp. clustered separately from all Laimaphelenchus species with available sequences in Genbank.

The L. liaoningensis n. sp. is the first report of a Laimaphelenchus species in China, and P. armandii is a new host for the genus.