Abstract: [Objective] Two formae speciales of Marssonina brunnea in Populus genus cannot be distinguished easily based on their morphologies and some nucleic acid molecular sequence marks. In this study, we applied combination of the phylogenetic trees with Marssonina ribosomal internal transcribed spacer(ITS)sequence and the secondary structures of ITS2 to discriminate the specificities of the two formae speciales. [Method] The twenty-three strains were cultured for fungal spore morphology observation. Genomic DNA was used as a template for PCR amplification on their ITS sequences. The reference ITS sequences of Marssonina were downloaded from the NCBI database.Three phylogenetic trees based on ML, MP and Bayes methods, respectively,were constructed.The ITS2 database was applied to predict ITS2 secondary structure model,and analyze the characterizations of the two formae speciales from the perspective of molecular evolution.[Result] The pathogen spore had a typical morphology of a forma specialis in host leaves in the field,but the spores were quite different in morphology on culture medium. We can't effectively distinguish two formae speciales on the basis of the traditional number of germinated germ tubes. Three kinds of ITS sequences phylogenetic trees according to ML, MP and Bayes approaches showed that there were three species in Marssonina genus. But reference ITS sequences with a formae speciales from M. brunnea can't be accurately located into the other same formae speciales with other strains. With the ITS2 secondary structure prediction model, it was found that Marssonina ITS2 secondary structure was identical in the three species, M. rosae, M. coronariae, and two formae speciales of M. brunnea. But the ITS2 secondary structures have multiple bases sites different among the three species. In the ITS2 secondary structure of M. brunnea leuce biotypes, the 17th base is A in Helix I, the 43th and 44th bases are AC in Helix Ⅱ. The compensatory base change(CBC) sites are the 76th C and 114th G, hemi-CBC site is the 75th U in Helix Ⅲ. In the ITS2 secondary structure of M. brunnea aigeiros biotypes, the 17th base is U in Helix I, the 43th and 44th bases are GU in Helix Ⅱ. The compensatory base change(CBC) sites are 76th U and 114th A, hemi-CBC site is the 75th C in Helix Ⅲ. These base changes are distinguished in two formae speciales in the ITS2 secondary structure.[Conclusion] The Marssonina strains can be distinguished in species level by three phylogenetic trees base on ITS sequences, but cannot be distinguished into subspecies; With comparative analysis of ITS2 secondary structures, the Marssonina genus is obviously different, and there are also compensatory base changes within formae speciales. Application of ITS2 secondary structures combined with phylogenetic trees can be more effective molecular characterizations to distinguish the two formae speciales in M. brunnea.

Key words: Marssonina brunnea, forma specialis, phylogenetic tree, the secondary structure of the ITS2, CBC analysis