Abstract:

【Objective】Parasitic nematodes are invertebrates that infest pine trees, feeding on plant cells and potentially harming tree health. Some nematodes can even kill pines (Pinus spp.). In 2022, a new parasitic nematode was isolated from black pine (Pinus thunbergii) and tentatively named the Liaoning nematode. To clarify its taxonomic status, both morphological and molecular biological identifications were performed to determine its species and guide its prevention and control.【Method】The Baermann funnel method was employed to separate nematodes from dead wood samples. After three washes with sterile water, the nematodes were transferred to Botrytis cinerea for seven days. Morphological characteristics of female and male adult nematodes were observed and measured using light microscopy. For molecular identification, DNA was amplified and sequenced using 18S rDNA, 28S rDNA, and COI gene primers to construct an evolutionary tree and further assess the taxonomic status.【Result】The primary morphological features of the Liaoning nematode are as follows: The average body length of female adults is 580.6 μm, mean body width is 17.3 μm. Female adults are slightly ventrally curved when heat-relaxed, with fine annules between the lip and body and a stylet with small basal swellings. The median bulb is ovoid, with a height and width of 11.8 and 8.4 μm, respectively. The vulva, without a vulval flap, is inclined at 45° and located between 60%-70% of the body length, with a distance from the posterior vaginal sac of 25.6 μm. The average distance between the vagina and anus is 131.9 μm. The tail measures 33.1 μm in length and has terminal shapes resembling snail feelers. The male nematode’s anterior region is similar to the female’s but forms a “J” shape when heat-relaxed and has a shorter body length, approximately 479.2 μm. The stylet length is 10.1 μm, and the body width is about 19.0 μm wider than that of female adults. The spicule length is 14.8 μm, with well-developed condylus and rostrum positioned in the middle and upper part of the spicules. The tail length is 28.0 μm, with short protrusions at the terminal. These morphological features align with A. paradanlianensis. DNA cloning and sequencing of the 18S rDNA, 28S rDNA, and COI genes produced target fragments of 914, 769 and 687 bp, respectively. BLAST alignment on NCBI revealed the highest similarity to A. paradanlianensis. Phylogenetic analysis indicated that the Liaoning nematode and A. paradanlianensis are closely related, confirming the nematode’s taxonomic status.【Conclusion】 Combining morphological and molecular characteristics, the Liaoning nematode isolated from P. thunbergii has been identified as A. paradanlianensis, marking its first discovery on pine trees in China. While morphological measurements of the Chinese population are slightly higher than those of the Korean population, the characteristics are generally consistent, possibly due to geographic variations or intra-species differences.

Key words: pine(Pinus spp.), parasitic nematodes, new record species, Aphelenchoides paradanlianensis