A nematode specie with a rare morphology was isolated from dead Pinus massoniana found in the Bocun Forest Farm, Yellow Mountain, Anhui Province, in 2017. A general observation using light microscope revealed that the population of the nematode was dominant and unique. The nematode was described and illustrated based on its morphological and molecular characteristics in order to identify its relationship with pine wilt symptoms. Besides, this identification of new nematode species would not only enhance the database of Aphelenchoides species, but also provide theoretical references for research on pine wilt disease.

Wood samples were sliced into 1 cm × 1 cm slices, followed by isolation of nematodes by the Baermann funnel method at room temperature for 8-12 h. A certain number of nematodes were rinsed with sterile water and then transferred into the Potato Dextrose Agar medium inoculated with Botrytis cinerea to acquire sufficient nematode population after incubation for 7 days at 25°C. Measurements, descriptions, and micrographs of nematodes were obtained using a light microscope and the data was statistically analyzed. The DNA of the nematodes was extracted and corresponding ITS, 18S and 28S D2-D3 regions were amplified, cloned and sequenced. A phylogenetic tree was constructed for subsequent molecular development analysis. The nematode species was identified by systematic morphological observation and statistical and comparative analysis of the molecular biological sequences.

The female nematode population was characterized by a body length of 642.0 μm, weakly annulated cuticle, rounded and offset lip region, and 10.7 μm long stylet with obvious small basal knobs. Additionally, the excretory pore was located anterior to the median bulb, 32.9 μm from the anterior end. The median bulb was rounded to oval, with conspicuous valve plates situated more or less centrally. The pharyngeal glands were long lobed, overlapping the intestine dorsally. The female reproductive system was highly developed and monodelphic, with the ovary outstretched forward and oocytes arranged into a single-row layout. Post-uterine sac was well-developed and 80.9 μm long in a pouch shape. The vulva was located in the posterior part of the body, with a simple slit visible in ventral view, and without a vulval flap. The tail was cylindrical and slightly ventrally arcuate, and the terminus was broadly rounded with a blunt peg. The general characteristics of the male nematode population were similar to those of the female; however, the males were usually shorter in length at 590.0 μm. The genital system of the males was monorchic, with the testis stretched forward, often extending to the vicinity of the posterior edge of the esophageal gland. The tail was conical, curved to the ventral surface, with a length that was about 3.0 times the diameter of cloaca. Three pairs of subventral caudal papillae were present in the male tail. Moreover, the males had paired spicules with rose-thorn shape and rounded apex and rostrum. Bursa and gubernaculum were absent. The neighbor-joining tree method based on ITS, 18S and 28S DNA sequences demonstrated that the nematode and A. rotundicaudatus are clustered in the same branch with 100% confidence.

Morphological and molecular analyses revealed the nematode species as A. rotundicaudatus. This is the first report of A. rotundicaudatus in China after its first introduction in the scientific literature. The Korean population mentioned in the original literature cannot reproduce on B. cinerea and only 46.0% males and 97.0% females were observed to have tails terminating in a blunt peg. In the Chinese population, nematodes could reproduce well on B. cinerea, and almost all the adults had their tails terminating in a blunt peg. Based on morphological and statistical analyses, the data of Chinese population is slightly larger than Korean population. However, the morphological characteristics are basically the same, and the variations detected could have been due to different geographic origins or intraspecific mutations.