First Report of Root-Knot Nematode, Meloidogyne graminicola, on Rice in Sichuan Province, Southwest China

Abstract

Root-knot nematode, Meloidogyne graminicola, is a major parasite of rice and causes substantial yield loss (Mantelin et al. 2017). In June 2018, stunned, yellowish plants were observed 30 days after transplantation in fields in Guanghan City (31°6′N, 104°13′E), Sichuan Province, Southwest China. Patches of seedlings with chlorosis were observed in source upland seedling nurseries. Galls and hooked tips were found in the roots of rice seedlings and plants. Rice plants (30) were randomly sampled from these and adjacent fields. Females and eggs were observed in galled roots and did not protrude through the root surface. Hatched second-stage juveniles (J2) were also found inside the galls. The perineal pattern of females (n = 9) was oval with low and round dorsal arches, smooth striae, sometimes with a few lines converging at either end of the vulva, and lateral lines obscure or absent. Measurements (mean ± SE, range) of J2 (n = 20): body length (446.7 ± 12.8 µm, 402.6 to 509.1µm), stylet length (12.1 ± 0.4 µm, 10.6 to 13.2 µm), tail length (70.2 ± 2.9 µm, 61.3 to 79.7 µm), and hyaline tail terminus (19.5 ± 1.0 µm, 16.5 to 22.7 µm); male (n = 10) abundant, body cylindrical and fairly long (1,270.0 ± 76.2 µm, 1,043.5 to 1,553.5 µm), stylet length (17.2 ± 0.7 µm, 15.2 to 18.9 µm), and spicule length (21.4 ± 0.4 µm, 20.1 to 22.0 µm). Morphometric analyses were consistent with M. graminicola (Golden and Birchfield 1965). Two individual females from each of 30 samples were transferred to tubes for DNA extraction. The D2 to D3 domain regions of 28S ribosomal RNA (rRNA) gene was amplified with the primers RK28SF (5′-CGGATAGAGTCGGCGTATC-3′) and MR (5′-AACCGCTTCGGACTTCCACCAG-3′) (Ye et al. 2015) and yielded a 589-bp PCR fragment. The internal transcribed spacer (ITS) region was amplified with species-specific primers Mg-F3 (5′-TTATCGCATCATTTTATTTG-3′) and Mg-R2 (5′-CGCTTTGTTAGAAAATGACCCT-3′) (Htay et al. 2016) and yielded a 338-bp PCR fragment. BLAST results of 28S rRNA gene and ITS region had 99% similarity with M. graminicola isolates available in GenBank. Sequence identities were further confirmed by Bayesian phylogeny analyses; they were nested in a well-supported clade (posterior probability = 1) together with other M. graminicola populations. The ITS and 28S rRNA sequences of the females from each of 30 samples were 99 to 100% identical. The ITS and 28S rRNA sequences of one population (GH-2) were deposited in GenBank (MK560173, 28S rRNA; MK559561, ITS). Morphological and molecular characterization confirmed this nematode as M. graminicola. To assess M. graminicola reproductive capacity, 2-week-old rice (Oryza sativa ‘Nipponbare’) seedlings were individually transplanted into a standard tube (25 × 95 mm) containing 10 ml of steam-sterilized sand and absorbent polymer substrate (Reversat et al. 1999), inoculated with 100 J2 of population GH-2 soon after transplanting, and then incubated in a growth chamber under 16/8-h light/dark at 26 to 28°C. There were 20 nematode-inoculated plants and 10 non-inoculated (tap water only) control plants. Twenty-five days after inoculation the number of egg masses and number of eggs per egg mass were determined and the multiplication factor [(number of egg masses × number of eggs per egg mass)/nematode inoculum level] calculated as 8.9 to 11.6. Hook-shaped and galled roots and stunned aboveground parts were observed on all inoculated plants; no symptoms were found on control plants. Observations confirmed the pathogenicity of the isolated M. graminicola population on rice. This is the first report of M. graminicola in Sichuan Province, a major rice-growing area. The first report of M. graminicola in China was in Fujian Province (Liu et al. 2011; Zhou et al. 2015), and it was soon found in other parts of China (Long et al. 2017; Tian et al. 2017; Wang et al. 2017). The infective J2 may be transmitted by gravity irrigation from field to field due to the topography of agricultural lands in the area. The extension of practices of upland nurseries, aerated irrigations, and direct seeding in Sichuan Province may further exacerbate the problem because such practices favor the survival and damage of M. graminicola (De Waele and Elsen 2007; Win et al. 2013). Further monitoring and research on control methods are needed.