First Report of Root-Knot Nematode <i>Meloidogyne incognita</i> Infecting <i>Siegesbeckia orientalis</i> (herba siegesbeckiae) in China

Siegesbeckia orientalis L. (Asteraceae) is a medicinal plant valued for its immunomodulatory, anti-inflammatory, anti-rheumatic, cardiovascular, antimicrobial, antioxidant, anti-tumor, and wound-healing properties (Wang et al. 2011). In September 2025, roots exhibited small galls with visible egg masses on the surface of S. orientalis plants in Enshi (30°54′76.02″ N, 109°90′46.05″ E; altitude 918 m), Hubei Province, China. Affected plants exhibited stunted growth and leaf yellowing, with approximately 20% of plants showing symptoms in the surveyed area. Nematodes were extracted from root samples by cutting the roots into 1-2 cm pieces and agitating them in water for 24 hours to allow the eggs and juveniles to hatch and disperse. Soil samples were processed using the Cobb sieving and decanting method followed by sugar flotation centrifugation to extract second-stage juveniles (J2s) (Barker 1985). Morphological characterization of adult females (n = 20) revealed the following measurements: body length (L) = 617.05 ± 52.68 µm, body width (W) = 385.46 ± 38.26 µm, and stylet length (ST) = 16.02 ± 0.75 µm. The perineal pattern of females displayed a high, trapezoidal dorsal arch with thick striations. Males (n = 10) had L = 1420.85 ± 200.35 μm, W = 35.46 ± 4.50 μm, ST = 23.56 ± 0.96 μm, and distance from the dorsal esophageal gland orifice to the base of the stylet (DGO) = 2.36 ± 0.15 μm. Second-stage juveniles (J2s; n = 20) had L = 398.02 ± 20.12 µm, W = 16.25 ± 1.35 µm, ST = 14.36 ± 1.02 µm, tail length (T) = 42.23 ± 4.56 µm, and hyaline tail terminus (HT) = 10.23 ± 2.12 µm. J2s also exhibited well-developed esophageal glands. These morphological traits were consistent with previous descriptions of Meloidogyne incognita Chitwood, 1949 (Kofoid &amp; White, 1919; Whitehead 1968). For molecular confirmation, genomic DNA was extracted from a single second-stage juvenile (J2). The V3–V5 region of the 18S rDNA gene and the D2–D3 region of the 28S rDNA gene were amplified using primers 18sf1/18sr1 (Waite et al. 2003) and MF/MR (Hu et al. 2011), respectively. The resulting 733 bp and 449 bp sequences (GenBank accession nos. PX588450 and PX588452, respectively) showed 100% and 98.22% identity with reference sequences MF177715 and KX752299, respectively, and were confirmed to be M. incognita by comparison with reference sequences. Pathogenicity tests were conducted by inoculating twenty healthy three-month-old S. orientalis seedlings grown in sterilized sand with 2,000 J2s per plant. Ten non-inoculated seedlings served as controls. After two months in a growth chamber maintained at 22°C, root galls and egg masses developed on all inoculated plants, matching field symptoms, while controls remained symptom-free. Females re-isolated from inoculated roots were confirmed as M. incognita using the species-specific primers Mi-F/Mi-R (Meng et al. 2004), which amplified an 850 bp fragment (GenBank accession nos. PX606139). The pathogenicity test was repeated three times under the same conditions to confirm the results. To our knowledge, this is the first report of M. incognita infecting S. orientalis in China. Growers should be informed of these findings to mitigate potential economic losses and implement appropriate management strategies.