| Abstract |
Salvia miltiorrhiza is a perennial herbaceous plant used in traditional Chinese medicine for hundreds of years to treat various diseases (Su et al. 2015). It is an important cash crop that is widely cultivated in southern Shaanxi province. In June 2021, in a field in Luonan County, Shaanxi, S. miltiorrhiza plants with stunting and leaf wilting symptoms were observed. The plants exhibited many globular galls on secondary and tertiary roots. Symptoms were typical of infection by root-knot nematodes (RKNs). Population densities of second-stage juveniles (J2s) ranged from 330 to 650 per 100 cm³. To identify the species, J2s and males were collected from soil in the root zone, and females were isolated from diseased roots. The perineal patterns of females (n = 12) were round, with low dorsal arches, obvious lateral lines, and characteristic small punctations near anus. Morphological measurements of females (n = 20) included body length (L) = 565.25 ± 33.9 (503.35 to 632.47) μm, body width (BW) = 420.00 ± 21.28 (378.27 to 452.51) μm, stylet = 11.11 ± 0.73 (10.05 to 12.29) μm, dorsal pharyngeal gland orifice to stylet base (DGO) = 4.69 ± 0.45 (3.82 to 5.32) μm, vulval slit length = 21.1 ± 1.33 (18.38 to 22.96) μm, and vulval slit to anus distance = 15.76 ± 1.24 (13.38 to 17.45) μm. The morphological characters of males (n = 7): L = 1,098.14 ± 82.99 (962.83 to 1,193.87) μm, BW = 28.44 ± 1.18 (26.59 to 29.83) μm, stylet = 18.27 ± 0.97 (16.57 to 19.28) μm, DGO = 4.89 ± 0.62 (3.82 to 5.68) μm, and spicule length = 24.04 ± 1.80 (21.30 to 26.71) μm. The key morphometrics of J2s: L = 380.24 ± 18.24 (354.43 to 423.13) μm, BW = 13.94 ± 0.70 (12.88 to 15.34) μm, stylet = 11.82 ± 0.49 (10.96 to 12.61) μm, DGO = 3.68 ± 0.42 (3.09 to 4.56) μm, tail length = 55.42 ± 5.81 (46.97 to 67.03) μm, and hyaline tail terminus = 13.79 ± 1.24 (12.0 to 16.51) μm. These characteristics are consistent with Meloidogyne hapla as described by Whitehead (1968). Ten females were put in 10 tubes for DNA extraction following Htay et al. (2016). Species-specific primers JMV1 (5′-GGATGGCGTGCTTTCAAC-3′) and JMV (5′-AAAAATCCCCTCGAAAAATCCACC-3′) were used for identification of M. hapla (Adam et al. 2007). A single 440-bp fragment was amplified by the primers, confirming the species as M. hapla. To confirm species identification, the ITS region was amplified using the primers 18S/26S (5′-TTGATTACGTCCCTGCCCTTT-3′/5′-TTTCACTCGCCGTTACTAAGG-3′) (Vrain et al. 1992). The ITS sequence was 768 bp (GenBank OM049198) and 100% identical to sequences of M. hapla (MT249016 and KJ572385). The mtDNA region between COII and the lRNA gene was amplified using primers C2F3 (5′-GGTCAATGTTCAGAAATTTGTGG-3′) and 1108 (5′-TACCTTTGACCAATCACGCT-3′) (Powers and Harris 1993). A 529-bp fragment was obtained and the sequence (OM055828) was 100% identical to that of M. hapla from Taiwan (KJ598134). Infection was tested in greenhouse conditions. Six 2-month-old S. miltiorrhiza plants were individually grown in 12-cm-diameter, 10-cm-deep plastic pots with sterilized soil and inoculated with 3,000 J2s hatched from collected M. hapla egg masses. Two noninoculated plants served as negative controls. After 60 days, inoculated plants had galled roots like those observed in the field. Many galls (61.33 ± 8.52) and egg masses (26.17 ± 4.79) were found on each root system. The nematode reproduction factor (final population/initial population) was 4.5. No symptoms were observed in controls. The nematode was reisolated from root tissue and identified as M. hapla with sequence-specific primers JMV1/JMV. These results confirmed that the population could infect S. miltiorrhiza. To our knowledge, this is the first known natural infection of S. miltiorrhiza with M. hapla in China. Including S. miltiorrhiza, the ingredients of many traditional Chinese herbal medicines were extracted from plant roots. RKN infection will seriously lower the quality of Chinese medicinal materials. Therefore, the RKN species should be identified in different Chinese herbal medicines.
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