Diagnosis and characterization of the ribosomal DNA-ITS of potato rot nematode(Ditylenchus destructor) populations from Chinese medicinal herbs

The potato rot nematode(Ditylenchus destructor) is a very economically important nematode in agronomic and horticultural plants worldwide. In this study, 43 populations of D. destructor were collected from different hosts across China, including 37 populations from Chinese herbal medicine plants. Obtained sequences of ITS-rDNA and D2–D3of 28S-rDNA genes of D. destructor were compared and analyzed. Nine types of significant length variations in ITS sequences were observed among all populations. The differences in ITS1 length were mainly caused by the presence of repetitive elements with substantial base substitutions. Reconstructions of ITS1 secondary structures showed that the minisatellites formed a stem structure. Ten haplotypes were observed in all populations based on mutations and variations of helix H9. Among them, 3 known haplotypes(A–C) were found in 7 populations isolated from potato,sweet potato, and Codonopsis pilosula, and 7 unique haplotypes were found in other 36 populations collected from C. pilosula and Angelica sinensis compared with 7 haplotypes(A–G) according to Subbotin' system. These unique haplotypes were different from haplotypes A–G, and we named them as haplotypes H–N. The present results showed that a total of 14 haplotypes(A–N) of ITS-rDNA have been found in D. destructor. Phylogenetic analyses of ITSrDNA and D2–D3 showed that all populations of D. destructor were clustered into two major clades: one clade only containing haplotype A from sweet potato and the other containing haplotypes B–N from other plants. For further verification, PCR-ITS-RFLP profiles were conducted on 7 new haplotypes. Collectively, our study suggests that D. destructor populations on Chinese medicinal materials are very different from those on other hosts and this work provides a paradigm for relevant researches.

甘肃省武威市荷兰豆根腐病病原菌种类的鉴定

【目的】明确武威市荷兰豆根腐病病原菌的种类,可为该病害的及时有效防治提供理论依据。【方法】采集荷兰豆根腐病株并分离和纯化病原菌,通过形态学特征结合ITS-rDNA与TEF-1α基因序列分析鉴定种类,并按照柯赫氏法则测定其致病性。【结果】分离得到16株Fusarium spp.,其中3株为燕麦镰孢菌Fusarium avenaceum,分离频率为18.75%;13株为茄镰孢菌F.solani,分离频率为81.25%,后者为优势种类。荷兰豆致病性试验症状表现与田间的基本一致,接种7 d后发病率为100%,病情指数为16.5~38.0,其中燕麦镰孢菌W14-1病情指数最高,为38.0;茄镰孢菌W8病情指数最低,为16.5。【结论】茄镰孢菌和燕麦镰孢菌是引起甘肃省武威市荷兰豆根腐病的病原,茄镰孢为优势病原。

甘肃省党参根结线虫病发生、分布及病原种类鉴定

为明确甘肃省党参根结线虫病的发生、分布及病原种类,在甘肃省党参主产区调查、统计田间根结线虫病发生情况,并通过形态学和分子生物学方法进行种类鉴定。结果表明,甘肃省6个党参主产区均有根结线虫病发生,病田率、病株率分别为40.7%、13.2%,病情指数为10.4。依据根结线虫各虫态的形态测量值和雌虫会阴花纹等特征,将危害党参的根结线虫初步鉴定为北方根结线虫Meloidogyne hapla Chitwood,1949。rDNA-ITS区段和28S rDNA D2D3区段序列比对和系统发育树显示,6个根结线虫群体无核苷酸差异,与多个北方根结线虫聚为一支且有较高的置信度。利用特异性引物Mh-F/Mh-R扩增得到北方根结线虫的特异性片段,大小为462 bp。故综合形态学和分子生物学特征,将危害甘肃省党参的根结线虫鉴定为北方根结线虫。

青海省当归茎线虫种类鉴定

采集和分离到青海省大通县、互助县4个当归茎线虫群体,通过形态特征和测量值比较,以贝叶斯法构建ITS-rDNA系统发育树,并用4种内切酶DdeⅠ、HinfⅠ、Tru9Ⅰ(MseⅠ)和SduⅠ进行ITS-RFLP分析。结果表明,青海省大通县、互助县当归茎线虫群体的形态特征与腐烂茎线虫(Ditylenchus destructor)基本一致,体长略小,雄虫尾长和c(体长/体宽)值略大,雌虫a(体长/尾长)值略大,其他测量值均与腐烂茎线虫模式种一致。通过rDNA-ITS序列系统发育分析,发现青海省大通县、互助县茎线虫群体与腐烂茎线虫群体聚为一支,但不能归为目前已知基因型。rDNA-ITS区段PCR-RFLP结果显示,青海省4个群体与已报道的腐烂茎线虫群体酶切结果不一致,且存在明显差异。经形态与分子生物学特征分析,将这4个线虫群体确定为腐烂茎线虫,表明该线虫已在青海省发生与分布。

腐烂茎线虫不同群体致病性差异分析

为明确不同来源腐烂茎线虫致病性差异,采用盆栽试验,对分离自马铃薯(DXP2、HLJP1和SXP1)、党参(TCC1)和当归(WYA3和HZA3)的6个群体进行致病性测定。每个群体分别接种马铃薯、甘薯和党参(500条/盆,3个重复),60d后统计病情级别和繁殖系数。结果表明,6个群体均可侵染马铃薯,4个群体可侵染甘薯,所有群体均未侵染党参。不同群体的致病性存在明显差异,马铃薯线虫群体对甘薯和马铃薯具有较强致病性,党参线虫群体可侵染马铃薯和甘薯,但病情级别和繁殖系数较低。当归线虫群体只侵染马铃薯,且致病性和繁殖系数极低。

甘肃党参中花生茎线虫的鉴定与记述

【目的】明确甘肃省渭源县党参Codonopsis pilosula病样中的茎线虫种类。【方法】采用形态学特征、ITSrDNA与28S-rDNA序列系统发育分析、特异性引物PCR扩增、PCR-ITS-RFLP相结合的方法进行种类鉴定。【结果】甘肃省党参茎线虫群体形态特征与花生茎线虫Ditylenchus arachis相似,形态测量均值虽存在差异,但是范围值基本一致;系统发育分析显示,该线虫与花生茎线虫聚为一支,特异性引物PCR扩增片段、PCR-ITSRFLP图谱均与花生茎线虫相同。【结论】结合形态特征与分子特征分析,将甘肃党参茎线虫群体鉴定为花生茎线虫,表明该线虫已在甘肃发生分布。