Species identification of Chinese herbal medicine Danshen root-knot nematodes in Linyi region, Shandong Province

To investigate the species of root knot nematodes (Meloidogyne spp.) in Chinese herbal medicines in Linyi region, Shandong Province, samples were collected from 6 diseased Danshen fields in Mengyin County and 1 diseased Danshen field in Pingyi County. Nematodes were isolated and the second-stage of juveniles (J2s) of root-knot nematodes were used for PCR amplification with the universal primer (rDNA-ITS region 194/195) and the species-specific PCR primers of root knot nematodes. PCR products and their sequences were used to determine the species of root-knot nematodes. Meanwhile, whether these nematodes may reproduce on tomato was also determined. The morphological characters of J2s and female perineal patterns of root-knot nematodes were identified to confirm the species.The results showed that the 194/195 universal primer pair in the ITS region amplified 750 bp products from all 7 samples, which proved to be root knot nematodes, and the identity among the obtained sequences ranged from 99.4% to 100%. As for six samples from Mengyin County, four samples belonged to M. arenaria and two samples belonged to M. incognita, respectively; and one sample from Pingyin County belonged to M. incognita; all samples sequences were 100% identical to those reported for M. arenaria and M. incognita, respectively. The identity of amplification products of the universal primer pairs reached 98.3% ~ 100% with those in GenBank for the two nematodes. All samples were able to propagate on tomato. The characteristics of J2s and female perineal patterns were correspondingly matched with those identified for M. arenaria and M. incognita.