Research On The Molecular Detection Method Of Several Important Forest Quarantine Pathogens

In order to further enhance the quarantine and inspection work on abroad logs and wood packaging and to provide efficient, rapid and accurate detection method for the quarantine departments, this paper do research on the detection of several important quarantine fungus of the entry forest by molecular method. In addition, we also establish a dangerous pathogen database of the entry forest. The main result is as below:1. The PCR detection of Ceratocystis fagacearum was establishedBased on the differences of the internal transcribed spacer (ITS) sequences of Ceratocystis spp., one pair of species-specific primers, CF01?CF02, was designed. The PCR product is 280-bp.To increase detection sensitivity, a nested PCR was developed by using ITS1?ITS4 as the first-round primers and CF01?CF02 in the second round, as it can detect 1 pg genomic DNA per 25-μl PCR reaction volume. More importantly, CF01 ?CF02 primers were successfully adapted to real-time PCR with a detection limit of 0.1 pg genomic DNA per 20-μl PCR reaction volume. Using these two methods, we could rapidly and accurately detect the pathogen in artificially infected wood and soil.2. Multiplex PCR detection of Ceratocystis fagacearum and Phytophthora ramorum was establishedUse the specific primers for Ceratocystis fagacearum CF01/CF02 and the specific primers for Phytophthora ramorum Phyto1/ Phyto4 designed by Haden et al.,2004,we established a Multiplex PCR detection system of these two pathogens.After PCR amplification, we got two bands of 687-bp and 280-bp. The detection sensitivity was 10 pg genomic DNA per 25-μl PCR reaction volume.3. The PCR detection of Inonotus weirii was establishedBased on the differences of the internal transcribed spacer (ITS) sequences of Inonotus spp., one pair of species-specific primers, IW1?IW2, was designed. The PCR product is 165-bp.The detection sensitivity was 100 pg genomic DNA per 25-μl PCR reaction volume. In addition,IW1 ?IW2 primers were successfμlly adapted to real-time PCR with a detection limit of 0.1 pg genomic DNA per 20-μl PCR reaction volume. Using these two methods, we could rapidly and accurately detect the pathogen in artificially infected wood.4. Multiplex PCR detection of Ophiostoma wageneri,Inonotus weiri and Fusarium circinatum was establishedUse the specific primers for Ophiostoma wageneri LEPTO1/LEPTO2 designed by Wolfgang et al.,2005 and the specific primers for Inonotus weiri IW1/ IW2 and the specific primers for Fusarium circinatum S1/S2 designed by TaiLing Liao et al.,2007,we established a Multiplex PCR detection system of these three pathogens. The detection sensitivity was 10 ng genomic DNA per 25-μl PCR reaction volume. Use this method,we successfully detected the Ophiostoma wageneri from a batch of USA entry wood packing.5. A dangerous pathogen database of the entry forest was establishedCombined with the varities of the entry forest and the dangerous pathogens they could be carried, we sumarried and collected about 70 species of dangerous pathogen's messages, which include all quarantine pest of entry forest in China qurantine pest directories. Then we established a dangerous pathogen database of the entry forest. The user could log in the website to retrieve the pathogens messages.