Studies On Taxonomy Of Rhytismataceae On Lithocarpus Spp. And ISSR Analysis Of Coccomyces And Hypoderma

Systematics of part of species of Rhytismataceae on Lithocarpus spp.was studied from morphology,development biology,ecology,distribution,culture characters etc.with the techniques of modern mycology.And phylogenetic relationships of intraspecies and similar interspecies of 23 Hypoderma strains and 12 Coccomyces ones were studied.It is clear further to the phylogenetic relationships among these groups.Some samples of Rhytismataceae on Lithocarpus spp.from the mountains of south and west Anhui and were systematically studied according to the taxonomic principles of Darker(1967),Korf(1973),Cannon & Minter(1983,1986),Johnston(1986),Spooner (1991),Kirk et al.(2001).7 species among 2 genera were identified.Among the taxa there were 2 new species including Lophodermium isodiametum sp.nov.,Coccomyces mucronatoides sp.nov.;The studies of 5 known species involved in adding characters of morphology,development biology,new record of host and geographical distribution. Morphological characteristics were described and illustrated,and hosts and distribution were recorded for all the species,and their similar groups,parasititism and nosogenesis were discussed.Dichotomous key of the species was compiled.The DNA of 12 Coccomyces strains and 23 Hypoderma ones were respectively extracted.The ISSR-PCR reaction system was optimized,and the DNA from intraspecies and interspecies were amplified.It is necessary to find a ISSR reaction system suitable for this experiment by the method of the single factor screening in order to promote the stability and repetition.The reaction system of Coccomyces and Hypoderma were optimized in the quantity of primers,template,Mg²⁺,dNTPs,Taq DNA polymerase and annealing temperature,annealing time,elongation and cycle numbers.The optimal ISSR-PCR reaction system included 2.5μl 10×Taq Buffer,8ng/μl template,0.15mmol/L dNTPs,9.0μl ddH₂O,2.5mmol/L Mg²⁺,1.0UTaq DNA polymerase,0.4μmol/L random primers in the system of 15μl.The amplification process as follow:94℃beforehand denaturalization for 2 minutes,35 cycles;94℃denaturalization for one minute, 48～54℃(determined according to the annealing temperture of different primer) annealing for 60 seconds,72℃elongation for 2.5 minutes,after the last circulation,then 72℃elongation for 10 minutes.The DAN of 12 strains of Coccomyces were amplified with 8 collected random primers by the optimal process,134 fragments were obtained,129 polymorphic ones among,accounting for 96.26%.The most sites,20 ones,was amplified by Primer S45.The least ones,13 ones,was amplified by Primer S13.The length of most amplified fragments ranged from 150bp to 2000bp.The phenotype was well consistent with the results of ISSR based on the clustering tree map which were established by the method of UPGMA.The DAN of 23 strains of Hypoderma were amplified with 8 collected random primers using the reaction process,134 fragments were obtained,127 polymorphic ones among them,accounting for 94.78%.The most sites,21 ones,were amplified by S42.Fifteen sites were the least,amplified by Primers S31,S32,S46.The length of most fragments ranged from 150bp to 2000bp,too.From the clustering tree map which were established by the method of UPGMA,23 strains were classified into 4 sorts at the genetic similarity coefficient of 0.58.By the synthetical comparison of ISSR analytical results and the phenotypic characteristics,the genetic difference of intraspecific strains of Hypoderma rubi was embodied in the size,shape and deep of ascomata,the shape of paraphyses,the size of asci and ascospores,and the difference of hosts and regions.The interspecific difference of Hypoderma is mainly embodied in morphological characteristics,host kinds and inhabiting position.