马铃薯腐烂茎线虫特异性分子检测技术研究  

Species specific molecular diagnosis of Ditylenchus destructor populations occurring in China

作　　者：宛菲[1] 彭德良[1] 杨玉文[1] 何月秋[2] 

机构地区：[1]中国农业科学院植物保护研究所植物病虫害国家重点实验室,北京100193 [2]云南农业大学农业生物多样性与病虫害控制教育部重点实验室,昆明650201

出　　处：《植物病理学报》2008年第3期263-270,共8页Acta Phytopathologica Sinica

基　　金：国家自然科学基金资助项目(30370930);国家“973”计划(2002CB111400);“十一五”国家科技支撑计划项目(2006BAD08A15;2006BAD08A14)

摘　　要：本研究利用通用引物(rDNA1/rDNA2)研究了21个国内甘薯茎线虫(Ditylenchus destructor)群体和1个韩国马铃薯茎线虫(D.destructor)群体的rDNA-ITS序列,从21个国内群体中扩增出2个大小不同的ITS片段,分别约为940 bp和1 100 bp;经克隆、序列测定和分析比对发现其ITS区存在特异性差异,分别命名为A型和B型,其中18个群体DdTH、DdCL、DdJN、DdMY1、DdYX1、DdZZ、DdLN、DdDX1、DdFN、DdYX2、DDSX1、DdDX2、DdXY、DdLL、DdSX2、DdLY、DdMY2和DdPY的ITS扩增产物约为940 bp,称之为A型马铃薯腐烂茎线虫(940 bp),3个群体DdSH,DdTS,DdYS为B型马铃薯腐烂茎线虫(1 100 bp)。设计构建并筛选出A型和B型马铃薯腐烂茎线虫2对特异性引物DdS1/DdS2和DdL1/DdL2,分别扩增出A型马铃薯腐烂茎线虫、B型马铃薯腐烂茎线虫群体的特异片段252 bp和485 bp;引入D3A/D3B作为内标,设计出一步双重PCR检测技术;同时优化了检测体系和PCR反应程序。该技术具有较高的特异性和灵敏性,能快速、准确地检测出不同型的马铃薯腐烂茎线虫群体。The amplification of rDNA-ITS region with the universal primers rDNA1, rDNA2 （rDNA1/ DNA2） from twenty-one stem nematode populations（Ditylenchus destructor）on sweet potato from China and one population on garlic from Korea yielded two different fragments （940 bp and 1 100 bp）, after cloning, sequence analyzing and aligment, It was found that there are two distinguish different sequences of amplification products of rDNA-ITS and clearly clarified ITS products into two different types, named Type A （940 bp） and Type B （ 1 100 bp） respectively, rDNA-ITS sequences of eighteen populations of sweet potato stem nema- tode（DdTH, DdCL, DdJN, DdMY1, DdYX1, DdZZ, DdLN, DdDX1, DdFN, DdYX2, DDSX1, DdDX2, DdXY, DdLL, DdSX2, DdLY, DdMY2, DdPY） belong to Type A, the ITS sequence is 940 bp, whereas the other three populations of DdSH, DdTS, DdYS belong to Type B, the ITS sequence is 1 130 bp. Sequence alignments of the ITS region of the twenty-one populations of D. destructor showed there is distinguish variation between Type A and Type B. Sequence alignments of the Type A population of D. destructor showed that the similarity were 99.6% ; the Type B populations had 98.28% similarity. There is high similarity （95.44%） between the Chinese populations of Type B and American population of D. destructor （AY987007）.Two specific primer pairs, DdS1/DdS2 and DdL1/DdL2 were designed and developed to detect the Type A and type B populations of D. destructor respectively. The specific amplification product for Type A with specific primers DdS1/DdS2 is 252 bp, and for type B with specific primers DdL1/DdL2 is 485 bp. A method for rapid molecular diagnosis of sweet potato stem nematode based on duplex PCR with specific primers and universal primers D3A and D3B was described. This study provided an accurate and effective method for quick detection of D. destructor.

关 键 词：马铃薯腐烂茎线虫 双重PCR 特异性引物 

分 类 号：S432.45[农业科学—植物病理学]