Studies On Detection Of Pine Wood Nematode By Real-Time Fluorecent PCR And Population Differentiation Of Sweet Potato Stem Nematode

1. Diagnosis of pine wood nematode, Bursaphelechus xylophilus, by real-timefluorescent PCR using TaqMan probe.Bursaphelenchus xylophilus has caused great damage to the forestry and ecologysystem in some countries of North American and East Asia and Europe. It is an importantpest of quarantine concern. It has been considered that B. xylophilus is the dominantpathogen of pine wilt disease but other Bursaphelenchus don't infect pines. So, it isimportant for the Entry-Exit Qurantine and forest protection to find a method which coulddiagnose the B. xylophilus fleetly and accurately. Comparing to the traditional PCR,real-time PCR make little pollution to the environment and works more conveniently andspeedy. In this study, a set of primers and one specific TaqMan probe for Pine WoodNematode, were designed by comparison of the sequence differences in rDNA-ITS regionbetween PWN and its seven morphologically similar species. A real-time fluorescentdiagnosis system, rapid, accurate and sensitive, has been constructed. With this method,1/20 content DNA of a nematode can be detected accurately.2. Studies on population differentiation of sweet potato stem nematode.Sweet potato stem nematode is a crucial disease in agriculture. Ditylenchus destructor has beenconsidered as the pathogen, which was firstly founded from potato; the nematode infectedpotato and resulted in rot. But the main host of D. destructor is sweet potato in China. 7groups of D. destructor, 2 groups of D. dipsaci and a group of D. myceliophagus were analysedby RFLP pattern in this study. The internal transcribed spacer regions of the 10 populations ofDitylenchus were analyzed using PCR-RFLP techniques. The result showed that 2 populations of D.destructor from Shandong province have unique enzyme-digestion pattern compared to otherpopulations of this species. The study also supports that PCR-ITS-RFLP method is a valued tool fordifferentiation of D. destructor, D. dipsaci and D. myceliophagus. Using online softwareclustalw1.82, 15 rDNA-ITS nucleotic sequences were aligned. The result showed that thesequence difference between D. destructor and D. myceliophagus is 13-31%, and 43-56% between D. dipsaci and D. destructor, and 50-51% between D. dipsaci and D.myceliophagus. Based on the alignment result, the phylogenetic tree of the above 3 specieswas constructed, which shows the similar results as sequence alignment. That is, D. dipsacidiverged out from more ancient ancestor firstly, and sequent divergence occurred betweenD. destructor and D. myceliophagus.