First Report of Southern Root-Knot Nematode, Meloidogyne incognita, Infecting Isatis indigotica Fortune in Anhui Province, China.

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Isatis indigotica Fortune, widely cultivated in China, is an important Chinese herbal medicine, mainly used to treat cold and fever. In October 2020, galls (Fig. 1), as many as 65 per root, were observed on the roots of I. indigotica in Taihe, Anhui Province, China (117°21'19.5"N, 32°57'59.5"E), and samples were taken. The infected plants were weak, and the leaves are wilting. Second-stage juveniles (J2s) were dissected from the egg masses released by females. Excretory pores of females were located nearby median bulb (Fig. 2A). The dorsal arch of the perineal pattern (n = 10) of the female was elliptical, and the dorsal arch was relatively high with smooth to wavy lines (Fig. 2B). Morphometrics of females (n=10): body length (L) = 595.5 ± 24.0 (570.0-620.5) μm, body width (W)= 350.5 ± 30.0 (320.0-390.5) μm, stylet length = 13.6 ± 0.7 (12.1-15.4) μm (Fig. 2A), and the distance from dorsal esophageal gland orifice to base of stylet (DGO) = 3.5 ± 0.2 (2.8-4.0) μm (Fig. 2B). J2s (n = 20) had the following characteristics: L = 383.2 ± 12.5 (337-430) μm (Fig. 2C), a = 22.0 ± 1.1 (20.3-24.4) μm, c = 8.4 ± 0.5 (7.5-10.5) μm, stylet length = 12.4 ± 1.5 (10.1-14.6) μm, DGO = 2.9 ± 0.6 (2.0-3.6) μm (Fig. 2D), tail length = 39.5 ±3.4 (32.0-48.5) μm and hyaline tail terminus = 10.5 ± 0.5 (9.5-11.2) μm (Fig.1E). There were four lines on the lateral field of J2s (Fig. 2F). Females and J2s obtained from galls had uniform morphological and molecular characteristics were confirmed to be Meloidogyne incognita. Live J2s were detected in all soil samples with a mean of 120 ± 15 J2s/100 ml of soil. Five 4-week-old I. indigotica plantlets, grown in pots (500cm3) with sterilized soil were inoculated with 1000 J2s from egg masses per pot and5 non-inoculated pots were used as control. Plants were well maintained under 25 ± 3°C in the greenhouse. Three plants were gently removed from the pots 30 days after inoculation, and an average of 50 galls per root was observed on the roots, and the resulting nematode reproduction factors (RF = final egg density ÷ 1,000, initial egg density) of 3.2, suggested that I. indigotica is a good host for M. incognita (Mojtahedi, 1988). There were no significant differences in main measurements and morphological characteristics between the Taihe population of M. incognita and that represented in "CIH descriptions of plant-parasitic nematodes" (Orton Williams, 1973). DNA was extracted from 5 single J2s, and ITS and 18S rDNA gene was amplified using the primer pair 18S/26S and 18s1.2a/18sr2b (Bernard et al. 2010; Vrain et al. 1992). The sequence of 18S rDNA (MW875892) was submitted to GenBank. Comparisons showed a sequence identity of greater than 99.8% for Meloidogyne incognita (MF177719.1). The rDNA sequences of M. incognita, M. hapla, M. javanica and M. arenaria are so homologous that rDNA-based differentiation is difficult. The SCAR primers can successfully distinguish M. incognita, M. hapla, M. javanica and M. arenaria. Five species-specific primer sets (Finc/Rinc; MORF-F/MTHIS-R; Jmv-F/Jmv-R; Far/Rar and Fjav/Rjav, Stanton et al. 1997; Wishart et al. 2002; Zijlstra et al. 2000) were used to species-specifically distinguish within the genus. The results (＋, ＋, －, －, －) proved that the Taihe population belonging to M. incognita. To our knowledge, this is the first report of M. incognita parasitizing I. indigotica. This finding may be important to medicinal plant industry, since M. incognita is one of the most harmful nematode pests in the world and would cause severe damage to I. indigotica.