First Report of Root-Knot Nematode Meloidogyne javanica Infecting the Medicinal Plant Polygonum multiflorum in China

Polygonum multiflorum Thunb. is a perennial herb of the Polygonaceae family. As a common traditional Chinese medicine, P. multiflorum is cultivated widely in the Southwest China. It is frequently used in clinical practices (Huang et al. 2013). On June 15, 2024, P. multiflorum plants exhibiting symptoms of stunting, leaf wilting, and multiple root galls associated with root-knot nematodes were detected in 15-hectare commercial fields near Kunming City (24°49'23″N, 102°51'2″E), Yunnan Province, China. Seventy-five percent of the P. multiflorum plants had root galls with an average gall index of 4.17. Random sampling of 100 seedlings from five fields indicated a combined infested area of about 0.1 hectares. Females and egg masses were manually picked out from galled tissues of P. multiflorum plants (n = 30) by using a fine pointed needle. Egg masses were placed on a filter paper to hatch for 3 d at 25ºC, and second-stage juveniles (J2s) were collected by method of Baermann funnel. The nematodes were identified through comprehensive morphological and molecular methods. Perineal patterns were rounded with low dorsal arch, trapezoid shape, smooth striae interrupted by a pair of incisures on both sides corresponding to lateral fields, clearly demarcated from striae by parallel lines, tail whorl often distinct. Females (n = 30): length 695.3 ± 60.4 μm, width 396.5 ± 38.4 μm, stylet length 15.7 ± 1.6 μm, and DGO length 4.02 ± 0.7 μm. J2 (n = 30): length 428.9 ± 8.7 μm, width 18.2 ± 1.6 μm, stylet length 13.8 ± 0.7 μm, DGO length 3.1 ± 0.4 μm, and hyaline tail terminus 12.6 ± 1.8 μm. These morphological characteristics were similar to the original descriptions of M. javanica (Rammah et al., 1990). DNA was extracted from a different single female (n = 10) and the specific sequences were amplified by PCR using the 18S rRNA gene universal primers 988F/1912R for Meloidogyne spp. (Holterman et al. 2006) and M. javanica-specific primers Fiav/Rjav (Adam et al. 2007), which generated 911 bp (GenBank accession no. PV274245) and 613 bp (PQ629528) fragments, respectively. Of the sequence PV274245, 908 bp was similar to the KP901063 (915 bp) of M. javanica with 99.23% identity (Yu et al., 2015), and 611 bp of the PQ629528 was matched to the KP411880 (612 bp) of M. javanica with 99.84% identity (Kaur et al., 2016). To verify the pathogenicity, individual seedling of P. multiflorum was transplanted into a plastic pot (15-cm-diameter × 10-cm-high) containing a sterilized mixed soil (humus soil/laterite soil/ perlite = 3:1:1), and infected with about 1000 J2s from the original population infecting P. multiflorum. Seedlings of P. multiflorum without inoculation of J2 was used as the negative control. All treatments had five replicates. The plants were grown in a greenhouse at approximately 14 to 26°C for 60 days. The pot experiment was repeated, in which the plants were allowed to grow for 60 days in a commercial field where we collected the nematodes. The inoculated plants whether in greenhouse or in the natural condition presented identical symptoms as observed in fields. The root gall rating was 3 with a reproductive factor of 1.8 in the greenhouse, and 2 with 1.4 in the natural condition according to the 0-to-5 RKNs rating scale (Huang et al. 2015). No symptoms or nematodes were observed on the control plants. The nematodes in pot experiments were reisolated and identified as M. javanica by the methods as described above. To our knowledge, this is the first report of M. javanica infecting P. multiflorum in China, thus further research would be necessary to develop control strategies.