The Studies Of Molecular Detection Technology For Several Quarantine Pests

Dutch elm blight, one of the most destructive plant diseases in the world, also known asDutch Elm Disease, mainly damages ulmus species. This disease hasn’t found in our country,but Ophiostoma ulmi has been included in the list of our country’s quarantine pest. In2009,Phenacoccus solenopsis was found in Guangdong province, this pest can make a great threatto cotton, so we should take pointed measures to prevent it from happening. In1998,Dysmicoccus neobrevipes was found in Taiwan, and this pest propagates and spreads speedly.In recent years, Dysmicoccus neobrevipes has damaged over10acres of sisal hemp plantarea.To enhance related port’s quarantine and inspection on imported plant disease and pest inour country and to make quarantine objects definite, this paper do research on susceptibilitywith Dutch elm blight of China’s native elm, and study molecular detection method for threekinds of quarantine pest and pathogen.The main results are listed as followed:1Pathogenicity of Ophiostoma ulmi in China’s elm.To compare pathogen of O. ulmi in China’s main elm species, in late April and early Maywhen disease happens easily, we use Ulmus davidiana Planch., Ulmus pumila, Ulmusdavidiana var. japonica, Ulmus szechuanica, Ulmus castaneifolia, Ulmus parvifolia and otherChina’s elm species as vaccination materials.The results show that all the nine elm speciesinfect this disease in the four research period. According to index of disease infection, Ulmuspumila is the most susceptible species, secondly are Ulmus davidiana Planch and Ulmusszechuanica, while the other species are less susceptible to this disease. After the diseaseshows effect on infected elms, we choose elms with symptom of this disease to re-separate thepathpgenic bacteria and use molecular method to identify it. By taking ad-vantage of ITSdifference area in O. ulmi and other species of Ophiostom, We design a couple of specificprimers OpF/OpR. The size of PCR product with the primers is304bp. We use it to identifythe pathogens of infected elms with symptom of this disease, and confirm the re-separatedpathogen is O. ulmi.2Research on PCR detection method of O. ulmi.Based on the differences of the internal transcribed spacer (ITS) sequences of O. ulmi,one pair of species-specific primers, OpF/OpR, was designed. The PCR product is304bp. Toincrease detection sensitivity, a nested PCR was developed by using ITS1/ITS4as thefirst-round primers and OpF/OpR in the second round, as it can detect1pg genomic DNA per25μL PCR reaction volume. We could rapidly and actually detect the pathogen.3The Real-time PCR detection of O. ulmi established. Based on the differences of the ITS sequences of O. ulmi, one pair of species-specificprimers, OpF/OpR, was designed. The PCR product is114bp. These specific primers weresuitable for Real-time PCR. Obtained with a higher detection sensitivity of this method, as itcan detect0.1pg genomic DNA per20μL PCR reaction volume.4Multiplex PCR detection of P. solenopsis and D. neobrevipes was established.Based on the differences of the COI sequences of Phenacoccus solenopsis, one pair ofspecies-specific primers, PTF/PTR, was designed. PCR product is358bp.Based on thedifferences of the COI sequences of one pair of species-specific primers, DnF/DnR, wasdesigned. PCR product is228bp. Using two pairs of specific primers, we established aMultiplex PCR detection system of these two pathogens. After PCR amplification, all can getpositive expansion. This method can be used to quickly and simultaneously detect theimported timber and carry P. solenopsis and D. neobrevipes.