First Report of Northern Root-Knot Nematode Meloidogyne hapla on Bupleurum chinensis in Gansu Province, China.

Bupleurum chinensis is an important traditional medicine with anti-inflammatory and immunomodulatory effects in China (Navarro et al. 2001). So far, the diseases reported on B. chinensis were caused by fungi (rust and root rot) and virus (Cucumber mosaic virus and Broad bean wilt virus 2) (Zhang et al. 2009). However, no diseases caused by nematodes were reported previously. Root-knot nematodes (Meloidogyne spp.) are one of the most destructive plant-parasitic nematodes with strong adaptability and diversity, infecting more than 5,500 plant species (Azevedo de Oliveira et al. 2018). In October 2020, symptoms of dwarf, leaf yellowing and roots with numerous knots on B. chinensis in several fields were observed in Dingxi City, Gansu Province, Northwest China (N 35°19'42″; E 104°2'24″). Subsequently, hundreds of eggs, mature males and females were exuded from dissection of washed root-knots. Morphological characteristics of females, males and J2s were examined under the optical microscope. The perineal patterns of females (n=15) were oval-shaped with a slightly dorsal arches, and the lateral lines and punctations on anus were observed in some specimens. Measurements (mean ± SD, range) of females(n=20): L (body length) = (525.23 ± 59.88 μm, 439.72 to 659.93 μm), W (maximum body width) = (403.92 ± 57.17 μm, 311.01 to 513.34 μm), St (stylet length) = (11.28 ± 1.05 μm, 9.82 to 12.91 μm), MBW (width of the median bulb) = (31.13 ± 3.32 μm, 23.66 to 35.55 μm), MB (distance from anterior end to center of median oesophageal bulb valve) = (64.45 ± 3.44 μm, 58,62 to 71.92 μm), and DGO (dorsal gland orifice to stylet) = (3.79 ± 0.60 μm, 2.72 to 5.00 μm). Male (n=20): L= (1038.25 ± 90.34 μm, 877.28 to 1206.12 μm), St= (18.13 ± 1.48 μm, 15.10 to 20.12 μm), a (body length divided by greatest body width) = (31.77 ± 4.03 μm, 23.29 to 41.16μm), MBW= (10.97 ± 0.78 μm, 9.05 to 12.31 μm), MB= (64.81 ± 3.45 μm, 59.59 to 71.38 μm), DGO= (4.05 ± 0.47 μm, 3.11 to 5.08 μm), and Spic (spicule length) = (22.57 ± 1.91 μm, 19.26 to 26.43 μm). J2 (n=25): L= (381.73 ± 25.85μm, 336.96 to 419.98 μm), St= (10.52 ± 1.03 μm, 9.15 to 12.14 μm), a= (24.35 ± 2.10 μm, 20.45 to 28.29 μm), DGO= (3.02 ± 0.42 μm, 2.42 to 3.79 μm), c (body length divided by tail length) = (8.90 ± 0.86 μm, 7.71 to 10.48 μm), and c' (tail length divided by body width at anus) = (4.18 ± 0.50 μm, 3.47 to 5.04 μm). According to morphological characteristics, root-knot nematode infecting B. chinensis was preliminarily identified as Meloidogyne hapla Chitwood, 1949 (Whitehead 1968). To further verify this result, DNA was extracted from ten individual females, the ITS region and the D2-D3 region of 28S rDNA were amplified using the primer TW81/AB28(GTTTCCGTAGGTGAACCTGC/ ATATGCTTAAGTTCAGCGGGT) (Subbotin et al. 2000) D2A/D3B (ACAAGTACCGTGAGGGAAAGTTG/ TCGGAAGGAACCAGCTACTA) (De Ley et al. 1999), respectively. PCR products were purified and sequenced. The sizes of ITS region and D2-D3 region of 28S rDNA were 557 bp and 762 bp, respectively. The sequence of ITS region (GenBank accession number: OK030559) was 99.46%-99.82% identical to the M. hapla from China (MT490918), New Zealand (JX465560), Australia (AF516722) and Japan (LC030357). The sequence of D2-D3 region of 28S rDNA (GenBank accession number: OK030558) was 99.58%-100.00% identical to the M. hapla from Canada (MW182329), Ethiopia (KJ645432), USA (KP901086) and China (MN446015). Furthermore, fragments obtained using the specific primers of M. hapla (Mh-F/Mh-R) were 462 bp, which also was consistent with that of M. hapla (Feng et al. 2008). Through morpho-molecular characterization, the root-knot nematodes on B. chinensis in China were identified as M. hapla. Six seedlings of B. chinensis were planted in 16 cm diameter, 20 cm deep plastic pots with sterilized soil in the greenhouse at 20-25℃ for pathogenicity test. After planted 21 days, 2000 J2s/pot were inoculated, six seedling uninoculated were used as control. After 90 days, all inoculated plants showed similar symptoms observed in the field, and nematode reproduction factor (final population density/initial population density) was 1.47. Meanwhile, no symptoms were observed on control plants. These results proved that the nematode infecting B. chinensis is M. hapla. To our knowledge, this is the first report of B. chinensis as a new host of M. hapla in China. Bupleurum chinensis is widely planted in Gansu Province, the plant species cultivated across an area of about 19.1 million hectares, accounting for 40% of the China's total output (Wang et al. 2017). The root system of B. chinensis infected M. hapla is stunned and short, seriously affect the quality of medicinal materials, and restrict the development of the local Chinese herbal medicine industry.