First Report of Root-Knot Nematode Meloidogyne hapla on Coptis chinensis in China.

Abstract

Coptis chinensis Franch. 1897, commonly known as "Huang Lian", is a widely cultivated flowering plant in the family Ranunculaceae, especially in China (Wang et al., 2019). Rhizoma copies, the dried rhizome of C. chinensis, is extensively used in traditional Chinese medicine due to its antibacterial, anti-inflammatory, antioxidant, antitumor, and other pharmacological properties (Gai et al., 2018). In September 2023, C. chinensis plants in Lichuan City (30°36'15.36" N; 108°59'36.45" E) exhibited several symptoms, including stunted growth with yellowish leaves and many galls on roots. We suspected that this damage was caused by root-knot nematode. Roots were taken to the lab and maintained at 10°C for morphological and molecular identification of root-knot nematodes and pathogenicity testing. Females, second-stage juveniles (J2), and egg masses were dissected and extracted from roots with galls. The perineal pattern of females was oval with a slightly flat dorsal arch in some groups and marked in the anus. Males had high head caps that were narrower than the head region, broadening beyond the first body annuli. Morphological measurements of males (n = 20) were as follows: body length (BL) = 1,171.68 ± 85.34 µm, maximum body width (BW) = 34.04 ± 2.69 µm, stylet length (ST) = 19.19 ± 0.46 µm, dorsal pharyngeal gland orifice to stylet base (DGO) = 4.27 ± 0.26 µm, and spicule length = 26.58 ± 1.34 µm. Measurements of females were as follows: BL = 900.26 ± 35.15 µm, BW = 594.84 ± 42.16 µm, ST = 14.76 ± 0.58 µm, DGO = 5.22 ± 0.48 µm, stylet median bulb width = 31.25 ± 2.95 μm. Measurements of J2 were as follows: BL = 420.32 ± 26.85 µm, BW = 14.86 ± 1.77 µm, ST = 11.96 ± 0.69 µm, DGO = 3.53 ± 0.51 µm, tail length = 73.05 ± 11.52 µm, and hyaline tail terminus = 11.26 ± 2.31 µm. These morphological characteristics were consistent with those of Meloidogyne hapla (Whitehead, 1968). For species identification, genomic DNA was extracted from 12 single J2. The sequence of the 5.8S-18S ribosomal RNA gene was amplified and sequenced with the universal primer 194/195 (5´-TTAACTTGCCAGATCGGACG-3´/5´-TCTAATGAGCCGTACGC-3´) for confirmation of Meloidogyne spp. (Qin et al., 2022). The 768 bp sequence (GenBank accession no. PQ284213) was 99.82% identical to the 5.8S-18S rDNA sequences of M. hapla (AJ421708). Then, the variable V3 and V5 regions of the 18S rDNA gene were amplified and sequenced with the primers 18sf1/18sr1 (5´-CGCAAATTACCCACTCTC-3´/5´AGTCAAATTAAGCCGCAG-3´) (Waite et al., 2003). The sequences for the target genes (PQ284215) showed 100% identity to sequences of M. hapla (OQ642146). To confirm the pathogenicity of the population, six one-year-old healthy C. chinensis seedlings cultured in sterilized sand were each inoculated with 2,000 J2s hatched from egg masses. Four noninoculated seedlings served as negative controls. After maintenance at 25°C for 90 days, galls appeared on the roots of inoculated plants consistent with the symptoms observed in the field, whereas the negative controls showed no symptoms. Females collected from inoculated plants were identified as M. hapla with the species-specific primer MH-F/MH-R (5´-GAATAGTCTCAACGTTTATC-3´/5´-ATGTGACAGCGAAAAGAATT-3´) (Fengt al., 2008), which amplified a fragment of 447 bp (PQ284214). This is the first report of C. chinensis being a host of M. hapla, which is crucial for developing and implementing management strategies to prevent the spread of the pest to other areas.