山东省临沂地区中草药丹参根结线虫的种类鉴定

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

为了调查山东省临沂地区中草药根结线虫(Meloidogyne spp.)的种类,对临沂地区的蒙阴县和平邑县7个发病丹参田采集丹参样品,并分离线虫,利用根结线虫的通用引物(rDNA-ITS区域194/195)和常见根结线虫种的特异性引物对二龄幼虫进行分子鉴定,对PCR产物及其序列比较分析进而确定线虫种类,同时确定这些线虫能否在番茄上繁殖,然后对确定线虫种类的二龄幼虫形态学和雌虫会阴花纹的特征进行了鉴定。结果表明,rDNA-ITS区域194/195通用引物对所有7个样品都扩增出750 bp产物,证明是根结线虫,序列之间的相似度范围是99.4%~100%;序列特异性引物扩增表明蒙阴县4个样品是花生根结线虫(M.arenaria),2个样品是南方根结线虫(M.incognita),平邑县1个样品是南方根结线虫。序列分别与已报道的花生根结线虫和南方根结线虫100%同源,通用引物扩增产物序列也分别与基因库中这两种线虫序列达到98.3%~100%相似性。所有样品线虫都能在番茄上繁殖,二龄幼虫的形态学特征和雌虫会阴花纹的特征与已鉴定的南方根结线虫和花生根结线虫的特征相同。

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.