First Report of <i>Meloidogyne hapla</i> Infecting <i>Polygonatum cyrtonema</i> in China

Polygonatum cyrtonema Hua is a perennial herb belonging to the Liliaceae family, widely distributed throughout the temperate Northern Hemisphere, including China (Zhao et al., 2018). As a traditional medicinal herb, the rhizome of P. cyrtonema exhibits numerous pharmacological applications and biological activities, conferring substantial economic and medicinal significance. In August 2024, stunted growth and chlorosis were observed in a commercial field of cultivated P. cyrtonema in Xuan'en County (30°5'49" N; 109°48'14" E; altitude 1560 m) , Hubei Province, China. Upon uprooting, the roots exhibited extensive galling and deformation, with numerous small, bead-like galls typical of root-knot nematode infection. Numerous egg masses of root-knot nematodes were observed on the fibrous roots, while the tubers remained asymptomatic. Soil samples collected from the rhizosphere of affected plants had a population density of 1,500 second-stage juveniles (J2s) per 500 cm3 of soil using the sieve-funnel technique (Hooper et al. 2005). Females and egg masses were hand-picked from dissected galls. Morphological measurements of males (n = 20) were as follows: body length (BL) = 1,245.60 ± 60.35 µm, maximum body width (BW) = 28.36 ± 2.84 µm, stylet length (ST) = 18.79 ± 0.36 µm, dorsal pharyngeal gland orifice to stylet base (DGO) = 4.58 ± 0.24 µm, and spicule length = 26.40 ± 1.27 µm. Measurements of females (n = 20) were as follows: BL = 798.20 ± 46.82 µm, BW = 487.18 ± 53.15 µm, ST = 14.76 ± 0.58 µm, DGO = 4.10 ± 0.56 µm, stylet median bulb width = 27.86 ± 3.02 μm. Measurements of J2 (n = 20) were as follows: BL = 395.34 ± 28.46 µm, BW = 14.62 ± 1.58 µm, ST = 13.86 ± 0.62 µm, DGO = 3.18 ± 0.59 µm, tail length = 70.81 ± 10.98 µm, and hyaline tail terminus = 12.35 ± 2.29 µm. These morphological characteristics were consistent with those of Meloidogyne hapla (Whitehead, 1968). J2s (n = 15) from each single egg mass were pooled (five J2s per sample) for molecular identification. The 5.8S–18S ribosomal RNA gene region was amplified and sequenced using the universal primer pair MF/MR (5´-TTAACTTGCCAGATCGGACG-3´/5´-TCTAATGAGCCGTACGC-3´) for molecular identification of Meloidogyne spp. (Qin et al., 2022). The resulting 481 bp sequence (PX219632) exhibited 99.79% identity with 99% coverage to the corresponding 5.8S–18S rDNA sequence of M. hapla (PV911583). Subsequently, the variable V3 and V5 regions of the 18S rDNA gene were amplified and sequenced using the primers 18sf1/18sr1 (5´-CGCAAATTACCCACTCTC-3´/5´AGTCAAATTAAGCCGCAG-3´) (Waite et al., 2003). The obtained sequences (PX218334) demonstrated 100% identity with 100% coverage to the corresponding regions of M. hapla (PQ284215). To confirm the pathogenicity of the population, a pathogenicity test was conducted twice. For each experiment, ten one-year-old healthy P. cyrtonema seedlings cultured in sterilized sand were each inoculated with 2,000 J2s hatched from egg masses. Six noninoculated seedlings served as negative controls in each experiment. After maintenance at 25°C for 90 days, galls appeared on the roots of all inoculated plants consistent with the symptoms observed in the field, whereas the negative controls showed no symptoms. Similar results were observed in both independent experiments. 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´) (Feng et al., 2008), which produced a 441 bp amplicon (PX229603). This study provides the first documented evidence that P. cyrtonema serves as a host for M. hapla. This finding is critical for the formulation and implementation of targeted management strategies to curb the spread of this nematode pest to non-affected regions.