First Report of Northern Root-Knot Nematode <i>Meloidogyne hapla</i> Infecting <i>Dipsacus asper</i> in Guizhou Province, China

Dipsacus asper is a perennial herb growing in the moist fields and mountains. The root of D. asper is an important traditional Chinese medicine known for its tonic, analgesic and anti-inflammatory properties (Xu et al., 2022). Owing to this medicinal value, it is cultivated as a cash crop. In June 2024, symptoms of root-knot nematode (RKN) infection, typically characterized by root galling, were observed on two-year-old plants of D. asper in a commercial field in Huaxi, Guizhou Province, China (26.38°N, 106.61°E). Aboveground parts of infected plants displayed stunted growth and yellowing leaves, while belowground symptoms included thinning main roots and small galls from which fibrous roots usually emerged and formed fibrous root clusters. Disease incidence ranged from 70% to 90% across five fields covering a total of 7 hectares. To identify the causal RKN species, males were extracted from the rhizosphere soil, females and egg masses were dissected from infected roots and second-stage juveniles (J2s) were obtained using a modified Baermann funnel method (Schindler 1961). Morphological examination revealed that adult females were pear-shaped; the perineal pattern was oval in shape with a low dorsal arch, fine striae, non-continuing lateral lines, and tail terminal punctations. Males exhibited a high head cap narrower than the head region and moderately developed stylet with weak knobs. Measurements (n = 20) were consistent with those of Meloidogyne hapla (Whitehead 1968): females – body length (BL) = 524.77 ± 23.60 μm, body width (BW) = 322.21 ± 13.48 μm, stylet length (SL) = 11.61 ± 0.44 μm, dorsal esophageal gland orifice to stylet base (DGO) = 4.38 ± 0.17 μm, vulval slit length = 18.46 ± 0.47 μm, and vulval-anus distance = 14.41 ± 0.43 μm; males – BL = 1334.18 ± 41.91 μm, BW = 35.56 ± 1.37 μm, SL = 18.79 ± 0.33 μm, DGO = 4.02 ± 0.30 μm, spicule length = 28.24 ± 1.13 μm; J2s – BL = 386.16 ± 4.61 μm, BW = 15.11 ± 0.29 μm, SL = 13.40 ± 0.20 μm, DGO = 4.07 ± 0.13 μm, tail length = 60.54 ± 1.50 μm, and hyaline tail terminus = 14.94 ± 0.38 μm. Molecular identification confirmed the morphological results. DNA extracted from 20 females produced a 442 bp product with species-specific primers JMV1/JMV2 (Adam et al. 2007). The D2–D3 region of 28S rRNA was amplified using D2A/D3B primers via bidirectional sequencing (De Ley et al. 1999), and the 762 bp sequence (GenBank Accession No. PQ589742) showed 100% identity to M. hapla (OM688337.1, MW690212.1). The ITS region was amplified with primer pairs 18S/26S via bidirectional sequencing and the sequence (No. PV134087, 768 bp) matched M. hapla (KJ572385.1, OQ269698.1) with 100% identity (Vrain et al. 1992). Pathogenicity was validated by inoculating ten nematode-free D. asper seedlings with 2,000 J2s per plant, which were directly extracted from the diseased field. Ten noninoculated plants served as controls. After 45 days under greenhouse conditions (25°C, 70% RH, 16h/8h light/dark), all inoculated plants exhibited leaf yellowing and root galling, while controls remained symptomless. The reproduction factor (RF = final population/initial population) was 9.35 (Chen et al. 2024). To our knowledge, this is the first report of M. hapla infecting D. asper in Guizhou Province, China. Given the plant's medicinal value and the high infection rate observed, accurate identification of the nematode is essential for developing effective management strategies in D. asper production systems.