First Report of <i>Meloidogyne arenaria</i> Parasitizing <i>Solanum muricatum</i> in Yunnan Province of China

Pepino (Solanum muricatum), a highly nutritious fruit, is cultivated commercially worldwide. In China, it is grown on approximately 80,000 hectares across regions including the Yunnan–Guizhou Plateau, Qinghai Plateau, and the Hexi Corridor (Yang et al. 2023). Root-knot nematodes (Meloidogyne spp.), particularly M. arenaria, have been reported to adversely affect pepino production in Turkey (Akyazi et al. 2012). In 2023, symptoms such as stunted growth and root galling were observed in pepino plants in Shilin County, Yunnan Province, China (24.7567888°N, 103.2717417°E) (Fig. 1A). Roots staining with erioglaucine disodium salt (Sunohara et al. 2020) revealed abundant egg masses on the root surface (Fig. 1B, C). Thirty individual egg masses were isolated under a dissecting microscope (Fig. 1D) and incubated at 28°C to obtain second-stage juveniles (J2s) (Fig. 1E). Adult females were also extracted from the roots and preserved in sterile distilled water at 4°C (Fig. 1F). Four days after hatching, J2s from each egg mass were pooled (five J2s per sample) for molecular identification to determine whether single or mixed Meloidogyne species were present. DNA extraction was performed following the method of Jiang et al. (2023), and PCR amplification was carried out as described by Mao et al. (2019). Universal primers 194/195, targeting the 5.8S-18S rDNA region (750 bp), confirmed the presence of root-knot nematodes. Species-specific primers were used to detect M. incognita (Finc/Rinc, 399 bp), M. hapla (Fhap/Rhap, 610 bp), M. javanica (Fjav/Rjav, 517 bp), M. naasi (Fnaa/Rnaa, 433 bp), M. arenaria (Fare/Rare, 420 bp), M. enterolobii (Fent/Rent, 322 bp), and M. chitwoodi (Fchi/Rchi, 800 bp). PCR amplification yielded a 750 bp product with the universal primer pair and a 420 bp product only with the M. arenaria-specific primer; no amplification was observed with other species-specific primers (Fig. 1G). This result was further confirmed using individual J2s from a single egg mass (Fig. 1H). Sequencing of the 194/195 amplicon (GenBank accession no. PV893697) showed 99.56% identity with M. arenaria (NCBI accession no. U42342.1). The species-specific amplicon (GenBank accession no. PV893698) exhibited 98.29-99.25% identity with other M. arenaria sequences available in GenBank (MW315990.1, PP236047.1, and MZ522722.1). These molecular results conclusively confirmed the presence of M. arenaria in pepino roots in Yunnan. To further characterize the nematode, morphological measurements were taken from J2s, females, and males. Morphological characteristics of J2s (n=20) were as follows: body length = 468.13 ± 10.91 µm (range: 389.21–536.48 µm); body width = 19.85 ± 0.91 µm (14.77–27.60 µm); stylet length = 15.81 ± 0.54 µm (8.28–17.71 µm); DGO = 6.02 ± 1.02 µm (3.34–9.76 µm); tail length = 50.53 ± 3.46 µm (40.15–64.27 µm); hyaline tail terminus = 14.93 ± 0.72 µm (11.75–20.76 µm). Female (n=20) morphometrics were: body length = 604.16 ± 54.71 µm (209.05–793.89 µm); body width = 428.41 ± 43.17 µm (120.14–632.48 µm); stylet length = 13.95 ± 0.33 µm (11.64–16.02 µm). Female perineal patterns exhibited a low, flattened to rounded dorsal arch, coarse striae ranging from smooth to slightly wavy, and dorsal and ventral striae intersecting at the lateral lines. The striae were slightly indented at the lateral lines, forming a shoulder on the arch, and the striae from the dorsal and ventral sides often meet at an angle (Fig. 2A), consistent with previous descriptions of M. arenaria (Xu et al. 2017). Males were obtained by inoculating J2s of this nematode onto soybean seedlings grown in seed germination pouches, which facilitates easier collection of males (You et al. 2018) (Fig. 2B, C). Morphological measurements of males (n=20) were as follows: body length = 1277.04 ± 30.61 µm (1108.78–1568.53 µm); body width = 42.99 ± 0.89 µm (34.24–50.35 µm); stylet length = 23.92 ± 0.33 µm (20.33–27.11 µm); DGO = 4.74 ± 0.27 µm (3.34–8.24 µm); copulatory spicule = 29.90 ± 0.87 µm (24.03–35.48 µm). To assess pathogenicity, three two-month-old healthy pepino seedlings were each inoculated with 1,500 J2s and maintained in a greenhouse (22–28°C, 16/8 h light/dark cycle). At 35 days post-inoculation, root systems were collected (Fig. 2D) and stained with erioglaucine disodium salt. Prominent galls and abundant egg masses were observed (Fig. 2E), with an average of 76 ± 6 (range: 54-93) egg masses per root system and 266 ± 21 (range: 209-347) eggs per egg mass. Confirmatory PCR using the M. arenaria-specific primer produced a 420 bp product, further supporting species identity (Fig. 2F). The symptoms observed matched those in field-collected plants (Fig. 1B, C), confirming the pathogenicity of M. arenaria to pepino. To our knowledge, this is the first report of M. arenaria parasitizing S. muricatum in Yunnan Province, China. Ongoing surveillance and research on the biology and distribution of M. arenaria in pepino will be essential for developing effective management strategies.