First report of North Root-Knot Nematode Meloidogyne hapla on Codonopsis pilosula in China

Worldwide, root-knot nematodes are among the most important plant-parasitic nematodes infecting and causing yield losses on many important plant species. They can cause extensive galling on plant roots, leading to serious economic losses (Azevedo de Oliveira et al. 2018). Codonopsis pilosula is used extensively in traditional Chinese herbal medicine and is a major economic crop in Gansu province. In the summer of 2019, a survey for plant-parasitic nematodes was conducted in Gansu province, northwest China. During the survey, 35 out of 76 fields of C. pilosula had plants exhibiting stunting, decline, and extensive galling on the secondary and tertiary roots. These are typical symptoms of infection by root-knot nematodes. Females, males, J2s, and eggs collected from roots of C. pilosula had the following morphometric characters: the perineal pattern of females (n = 10) were round-shaped, with low dorsal arches and small punctations near the anus, lateral line marked. Females (n = 20): body length (L) = 579.06 ± 50.19 μm (511.42 to 677.41 μm), maximum body width (W) = 423.81 ± 29.11 μm (396.72 to 467.41 μm), stylet length = 9.93 ± 0.88 μm (8.71 to 11.72 μm), distance from dorsal esophageal gland opening to the stylet knot (DGO) = 4.93 ± 0.42 μm (3.91 to 5.84 μm), stylet median bulb width (MBW) = 29.67 ± 3.61 μm (24.41 to 35.31 μm). Males (n = 10): L = 1,021.75 ± 106.38 μm (885.09 to 1,230.52 μm), stylet length = 17.15 ± 1.41 μm (15.27 to 19.89 μm), DGO = 5.04 ± 0.61 μm (4.24 to 5.83 μm), MBW = 9.82 ± 2.09 μm (7.11 to 14.24 μm), spicule length = 21.97 ± 1.67 μm (19.54 to 24.59 μm). J2s (n = 20): L = 363.66 ± 25.96 μm (322.41 to 398.88 μm), a = 24.41 ± 4.32 (21.33 to 27.12), stylet length = 10.41 ± 0.79 μm (9.01 to 11.32 μm), DGO = 4.01 ± 0.56 μm (3.22 to 5.13 μm), tail length = 53.15 ± 10.56 μm (32.74 to 71.24 μm). Eggs (n = 20): L = 73.92 ± 4.35 μm (68.43 to 80.13 μm), W = 25.19 ± 3.94 μm (20.48 to 30.74 μm). These morphological characteristics are consistent with Meloidogyne hapla Chitwood, 1949 as described by Whitehead (1968). Molecular analysis was conducted for further research; DNA was isolated from four individual females, and then the ITS region and D2/D3 region of 28S were amplified with primers18S/26S (Vrain et al.1992) and D2A/D3B (Al-Banna et al. 2004), respectively. The D2/D3 region of 28S sequence and ITS sequence were deposited in GenBank under accession numbers MN752204 and MN752202, respectively. D2/D3 region of 28S sequence was 99.84 to 99.91% identical to those of M. hapla from China (KJ755187), Myanmar (MN446015), and Japan (KF430798). The ITS region sequence was 99.31 to 99.61% identical to several sequences of M. hapla from GenBank, including ones from Spain (MH011978), Taiwan (KJ572385), the United States (AY268108), and Japan (LC030357). Furthermore, species identification was also confirmed using PCR to amplify with M. hapla-specific primers Mh-F/Mh-R (Feng et al. 2008). An expected PCR fragment of approximately 462 bp was obtained, which was consistent with M. hapla reported. No amplification products were observed in the control. A pathogenicity test of this M. hapla population was carried out in a growth chamber maintained at 20 to 25°C. Eight healthy C. pilosula seedlings were transplanted into plastic pots (one plant per pot). After 2 weeks, the roots of six plants were inoculated with 3,000 J2s and eggs per root system, and two uninoculated plants were used as a control. After 65 days, all inoculated plants exhibited yellowish leaves and stunting and had extensive galling on roots, which resembled those on field-grown plants. No galling or symptoms were observed on control plants. The final population density was quantified, and mean nematode reproduction factor (final population/initial population) was 4.3. These results confirm this root-knot nematode population on C. pilosula is M. hapla. To our knowledge, this is the first report of M. hapla in Gansu province and the first report of C. pilosula as a new host of M. hapla.