Studies On Taxonomy Of Rhytismataceae On Some Broad-Leaved Trees And ISSR Analysis Of Lophodermium

Some members of three genera in Rhytismataceae which collected from the south mountains of Anhui province were systematically studied with the most novelty and advanced poly-character integrated taxonomy method which is based on the characters of morphology,developmental biology,ecology and distribution.Fifteen Lophodermium strains were charaterized by ISSR-PCR reaction with 9 random primers whose gel electroersis diagrams can be amplified repeatedly and clearly.By the comparison of ISSR analytic results and the phenotypic characters,indicated the importance of some morphological characters such as the embedded position of ascomata,stromata,asci, ascospores,paraphyses and the category of hosts on the classification of species level. Relationships of some intraspecific and interspecific strains of Lophodermuim on angiosperms were affirmed.The main result is as follows:Some samples from the south mountains of Anhui province and Anhui Agricultural University Forest Protection(AUUFP) systematically studied with the techniques of modern classification.Morphology structure and development biology of these fungi were roundly examined according to taxonomic principles of Darker(1967),Kor(1973),Cannon & Minter(1986),Kirk et al.(2001).Among them,Coccomyces clavatus sp.nov,and Lophodermiun acutisporum sp.nov.were new species,and 6 known species of China were supplemented and studied in new hosts and new geography distribution.The living materials of fungi of Rytismataceae from Tiantangzhai National Forest Park were separated and cultivated by tissue isolation techniques.Eight strains of Coccomyces,Hypoderma rubi,Lophodermium and Rhytisma were obtained.The strains were cultured on MA culture medium,and then regularly observed and noted the shape, size,luster,structure,mycelium,the rate of growth,and if which produce pigment and conidiomata.Template DNA of 15 Lophodermium strains from Tiantangzhai National Forest Park were extracted by Benzyl Chloride.The ISSR-PCR amplified system was optimized from aspects of the dosage of template DNA,prime,Mg²⁺,dNTPs and Taq enzyme,and anneal temperature and time,elongation temperature and time,cycle number.The amplified diagrams were quite well.The final reaction system(15μl) included ddH₂O 8.8μl, 10×Buffer 1.5,Mg²⁺(25mmol.L^-1) 1.5μl,dNTPs(10mmol.L^-1) 0.3μl,primer(10umol.L^-1) 0.6ul,Taq enzyme(5U.μl^-1)0.3μl,template DNA(45ng.μl^-1) 2μl.Its amplified procedure was pre-denature at 94℃for 2min.cycles 35 times,and every cycle included denature at 94℃for 1min,anneal for 60s,elongation at 72℃for 150s.And the latest cycle elongation time is 10min.The DAN of 15 strains of Lophodermium were amplified with 9 collected primers by the optimal ISSR-PCR process,154 fragments were obtained,150 polymorphic ones among them,accounting for 97.4%.The most sites,21 ones,were amplified by S45,the second ones were 20.Fourteen sites were the least,amplified by Primers S12.The length of most fragments ranged from 100bp to 2000bp,too.From the clustering tree map which were established by the method of UPGMA.The result demonstrates that 15 Lophodermuim strains on angiosperms are clustered into 6 groups in genetic distance 7.08. By the comparison of ISSR analytic results and the phenotypic character,we can confirm the importance of some morphological character such as the embedded position of ascomata,stromata,asci,ascospores,paraphyses and the category of hosts on the classification of Lophodermium.