|
This paper studies Pathogen biological characteristics and PCR of two kinds of quarantine diseases white rust and black stem. Determined Pathogen biological characteristics. To establish conventional and real-time PCR assays method for detecting two kinds of Pathogen.White rust occur on the leaves in the form of white blisters, its diameter is 1～10 mm, the diameter of the spores heap leaves is 0.5～4 mm. By the biopsy examination, sporangium are spherical, oval or polygonal, single cell, colorless,16.32～21.18μm×15.12-20.54μm. Oospores are brown to black, walls of spikes, grow in the host organization, the size of 46.57-66.82μm×45.49～65.77μm, in general, sexual state will have been found by the end of July to early August.Using fungal universal primers NL1/NL4 on the PCR amplification sunflower white rust. The PCR products were gel electrophoresis, cloned, ligation, transformation, plasmid construction, restriction analysis and sequenced, these fragments were sequenced and compared the homology array in GenBank. Primers ATHP3/ATHP4、ATHF/ATHR and TaqMan probe ATH-X were designed based on the unique sequence of the strains. To establish nest PCR and real-time PCR assays method for detecting, To fill the gaps in the country and abroad.Samples were collected from stands of diseased sunflowers,pathogenic morphology, pathogenicity testing,and ribosomal DNA intergenic transcribed spacer(rDNA-ITS) sequence analysis, Using 10 kinds of restriction enzymes areas of ITS amplified products were digested with different restriction enzymes sites. Results revealed that the pathogen causing the sunflower disease of was Phoma helianthi Boerma. Strain in the PDA, HLA good growth medium. It can make full use of soluble starch and potassium nitrate. Optimum growth temperature is 24～28℃. Optimum pH is 5.0～7.0. Culture conditions in full sunlight will help the growth of mycelium. Regard the black stem and germ parts of DNA-ITS region nucleotide sequence as the target sequence, the black stem specific primers LEPB bacteria and LEPF, amplified fragment 420bp, which has been designed successfully. Thus, we can accurately distinguish black stem bacteria from other phoma bacteria. At the same time, this study is on the black stem of the sunflower for the ITS sequences of pathogen detection primers and probes. Then, a black stem against the bacteria specific primers for LEP1, LEP2 and the TaqMan probe PMB has been successfully designed that.
|