Study On Technology Of Fast-Molecule Detection For Pathogen Of Pine Needle Cast

Pine Needle Cast is a serious disease of needles on pine, which caused by the Lophodermium pinastri. And since MeiFeng Zhu reported this pathogen in 1927, it has been accepted by most of scholars studying in this field.This experimentation has designed some specific primers for the ITS region of the Lophodermium pinastri. And therefore we can quarantine the pathogen before the disease breaks out and spreads.So thatit can provide some suggestion for the subsequent prevention and cure.This experimentation collected the ill pine needles in the forestry centre of Mount-ErLang during July to August in 2008.And we isolated the Lophodermium pinastri using the method of ascocarp isolation.Then the DNA was extracted to expand the ITS region and compared in NCBI,so that we can offer the standard sequence for the specific primer design.The sequence of the specific primer named 5-3 is: F(5'-3'):5-AAGAATACAGGCTTCACG-3 18bp R(5'-3'):5-GGTCAACCTTGTATGGTG-3 18bpWhen extracting the total DNA of the pine needles,we compared 7 methods for it,such as the Nucleus Deposition,the improved Nucleus Deposition,the Secondary Deposition,the CTAB,the High Mineral Low pH,the Benzyl Chloride, the Urea.But only the Nucleus Deposition and the improved Nucleus Deposition worked well.Meanwhile,we concluded that the best condition for the PCR detection.That was:The annealing temperature 50℃-54℃,The primer density 0.2μL(10mM),The DNA template 1μL(20-30ng).Before March 2009 the Lophodermium pinastri was not detected all along.But from March 2009 (including it) we detected the Lophodermium pinastri obviously only in the cusp and central section.Then we sequenced the production of PCR(48) and the glue recycles(27) by the Sangon.The results were the same as the sequence of Lophodermium pinastri in NCBI.According to this experimentation,it is available to detect the Lophodermium pinastri using the PCR method during its latent period.Because the ITS region keep a high guard among various funguses and abundant diversification for itself. So that we can design specific primer to amplify ITS region of the Lophodermium pinastri without amplifying other epiphites.And if we sprinkle some antiseptics,burn down the ill needles and strengthen fertilization before the disease appears about two months earlier,especially when the little brown spots show up,we are able to prevent the growth and spread of Lophodermium pinastri...