Studies On Separation Purification Of The Pathogen Of Gentian Leaf Spot And Identification Systematically

To provide the precise etiological agent for the specifical preventation and curation of Gentian leaf spot, and meantime to improve the preventation and curation of the chemi-presticide and traditional Chinese medicine elevating the output in the unit area,on the base of the predecessor'research, this experiment studies and identified systematically the pathogen of Gentian leaf spot in Hei Longjiang province. In the course of this experiment, we verified systematically 44 pathogenic bacterium of the Gentian leaf spot separated and purified in 4 culturing rough gentian temitory-the Hei Longjiang province BEIAN CITY ZHAOGUANG FARM, QITAIHE BEIFANG CHINESE HERB RESEARCH INSTITUTE, JIAMUSI CITY LIANJIANGKOU FARM, and the medicinal plant in HLJ TCM UNIVERSITY by the traditional morphology identification identificationt technique, Gas chromatography with Mass spectrometry(GC-MS) Cellular fatty acids(CFAs) cluster analysis technique and ISSR molecular biology marker technique.At first, the gentian leaf blades with spot blotch from four places above were collected on five-points-method,and observe and record the whole disorder symptom. Four places gentiana leaf blades were separated and purified by Tissue Separation and Spore Suspension, 15, 9, 12, 8 strains were acquired respectively, and the 44 strains were identified by traditional morphology identification of symptom observation, pycnidium morphous size, conidia created form in macroscopy and pycnidia, morphous of generator cell, morphous of spore,length size of spore septum amounts in conidia properties microscopy.By the syudy and identification on the pathogen of Gentian leaf spot all over the world and morphology observation on the separated pathogenic bacterium, the result was yielded: 44 separated strains are not significant difference in morphology,and are the same genus according to mycetes classification key, namely Septoria microspora Speg in Septoria mycetesSphaeropsidales Adelomycete Subphyla.Secondly, because the pathogen of gentian leaf spot belong to Sphaeropsidales fungus category,at present traditional morphology is deficient of the identification classification ,so it is difficult to definite if pathogen of gentian leaf spot exist variation. Therefore, analysis and identification of gentian leaf spot pathogens with their cellular Fatty Acids is further carrried out by GC-MS. The strain samples were cellular fatty acids transmethylated with the NaOH-Methanol processing method, selection spectrum peaks after detected by GC-MS. Each relative peak area is calculated by percentage area normalization method so that the clustering analytical method manages in these data. The 44 strains pathogens are divided into three kinds through cluster analysis. In addition, according to international microorganism nomenclature, these kinds are denominated corresponding specific name on the base of preliminary traditional morphology identification. Bei-11022 and Qi-91018 is a same kind (d=25), its naming is Septoria microspora (speg) vai.Wxj.;Bei-51022 and Qi-31022 is same(d=7), its naming is Septoria microspora (speg) var.Wsf.;The other are same(d<4),it is Septoria microspora speg. now.Then,twenty primers synthesized based on oligonucleotide repetitive sequence, 36 bands were amplified by six primers were screened with the DNA from 44 strains.The results showed that the pathogen gentian leaf spot DNA templates are extracted higher quality after improved CTAB method;the optimal ISSR reaction system comprised l.Oul template DNA, 2.0ul ISSR primer> 1.2ul Mg2+, 0.4ul dNTP, 0.5 ul Taq DNA-polymerase, 2.0ul Buffer> 12.2ul H2O in a total volume of 20ul reaction mixture;with the procedure of one cycle denaturing at 94 °C for 5 min,40 cycles each involved denaturing at 94 °C for 30s,annealing at 50°C for 45s,extending at 72 °C for 2 min,a final estension at 72 °C for 6 min after the last cycle. The genetic distance and genetic resemblance wereanalyzed by PopGen32 software, the gentian leaf spot pathogen dendrogram of genetic distance is constructed by MEGA2 software. The cluster analysis result showed that 44 pathogen strains were formed three groups at 0.26 level :Bei-51022> Bei-41022. Lian-31207> Qi-31022. Qi-71022> Lian-11022> Qi-21207 ^ Bei-31105^ Xiao-101106 the nine strains were formed one group;Bei-11022> Qi-91018> Lian-21022> ■Bei-101001 > Lian-21106> Qi-101022 > Qi-51001 > Lian-71106 > Lian-61207> Qi-41105 the ten strains were formed another group;the others 25 strains were formed one group same as standard strain. It is thus evident that the difference individuals by ISSR genetic marker cluster analysis obviously exceed those by GC-MS cluster analysis,but their classification tendency is same on the whole. The ISSR genetic marker further conforms variation information of GC-MS cluster analysis in the identification and classification of the separation 44 strains.Eventually, the ultimate conclusion is taken from the three methods above entire aspects: The Bei-51022 and Qi-31022 two strains, the Bei-11022 and Qi-91018 strains are two different kinds,but they are two variations or subspecies from Septoria microspora Speg.The Bei-11022 kind is named Septoria microspora (speg) var.Wxj.-, the Bei-51022 kind is named Septoria microspora (speg) var. Wsf.