Development And Application Of Rapid Molecular Detection Methods For The Dwarf Mistletoe (Arceuthobium Sichuanense)

Arceuthobium sichuanense is a perennial parasitic seed plant that parasitizes Picea crassifolia,has become devastating diseases to the spruce forest of three river source region. Its great damage, long incubation period and complex infection characteristics hinders the prevention and cure of A.sichuanense. In order to achieve early diagnosis and confirm the the system infection characteristics of A sichuanense, providing technical support to the seedling andcultivation of spruce and prevention of A.sichuanense, we developed high specific, sensitive and rapid method based on Real-Time PCR and loop-mediated isothermal amplification (LAMP), and the field samples were tested.1、Comparing the sequence of A.sichuanense and host Picea crassifolia, the specific primers to A.sichuanense were desigened based on Real-Time PCR and LAMP, and the systems of two methods were optimizated to A.sichuanense. Real-Time PCR is an automation technology, it can effectively avoid the pollution. LAMP is simple to operate, needs no special equipment, and the results could be judged according to the hydroxy naphthol blue (HNB) the chromogenic reaction. It is more suitable for for the generalization.2、In order to detect Real-Time PCR and LAMP primers specificity, the DNA of A.sichuanense and host Picea crassifolia were amplified, and the results show that, primers specificity of two methods are relatively good. Detecting the primers sensitivity, and compared with conventional PCR. It turned out that, the conventional PCR is lowest with 10-1ng/μL, LAMP is secend with 10-2ng/μL, and Real-Time PCR is highest,which is 10-4ng/μL.3、Testing the different infected branches using the established Real-Time PCR and LAMP. A.sichuanense was tested in asymptomat branches,showed that two methods can be used to the early diagnosis of A.sichuanense.The result of testing to buds showed that the endophytic system can extends to the buds of the host, indicating that A.sichuanense endophytic system exhibits a highly specialized parasitic strategy known as isophasic parasitism.Different branches were tested, the reuslt showed that A.sichuanense endophytic system can expand in spruce branches, confirming the the system infection characteristics of A.sichuanense.