| To confirm the phylogenetic placement and diversity of this kind fungus on Populus spp. inChina, extensive collection of Cytospora spp. and their teleomorph samples (Valsa spp.,Leucostoma spp., Valseutella spp., Valsella spp.) were gathered from Populus in China,identification and phylogeny of isolate were performed by morphological characteristicsincluding locule forms (Cytospora spp.) and stroma features (Valsa spp.) and DNA sequencehomology of the ITS-rDNA and β-tubulin partial sequences, furthermore, the genetic diversityof Cytospora chrysosperma was studyed by sequence analysis and RAPD marker. It is hopedthat to screen disease-resistant poplar varieties and create a new clue in control strategies forthis kind of pathogen in the long term.1Samples of poplar stems and branches with canker symptoms were collected fromcultivated poplar factions including Leuce, Tacamahaca, Aigeiros, which from in the mainpoplar-producing regions of China, including Heilongjiang, Jilin, Liaoning, Inner Mongolia,Henan, Sichuan provinces as well as2municipalities of Beijing and Chongqing from2011to2012.276canker specimens were obtained, and128of these were of seriously infected byCytospora spp, strains were isolated from lesions of infected stems or branches. In total,249Cytospora spp. strains were isolated from Populus from the main poplar producing regions inChina.2Isolates were identified based on morphology and DNA sequence homology of ITS-rDNAand β-tubulin. Microscopic structures of the fungi were examined on natural subtrates and inculture media by using the latest descriptive terms of locule forms. We found4conidiomatatypes: rosette cytosporoid, labyrinthine cytosporiod, Leucocytosporoid, torselliod. Isolatesreference were compared with the reference strains collected in other parts of the world,phylogenesis was performed in PAUP*4.0b10and Mrbayes using method of MP and BI.33representative genetically distinct sequences from249sequences of isolate residing within thepopulations of5known species and2unique lineages.1of them was described as new:Cytospora davidiana Y.L.Wang&X Y Zhang&Q. Lv, sp. nov. C. atrocirrhata and C.kantschavelii represented new reports from China, in addition,3of them corresponded with theprevious morphology-based taxa: C. chrysosperma, C. translucens, C. fugax, and remaining1phylogenetic species Cytospora sp.1was not nominated accurately due to the paucity of thematerials. Moreover, poplar and willow was found be new hosts of C. translucens in China, andC. chrysosperma was of a dominant species accounting for82%of the strains obtained in inthis study.Description of Cytospora davidiana sp. nov.: Conidiomatal stromata immersed in bark,erumpent, ovoid to elongate cylindrical,0.2-1.2cm up to1.5cm diam. Discs dark brown,nearly flat, circular to lenticular,0.1-2.0mm diam,1-10ostiole(s). Ostiole, dark grey, oftenabove the disc surfaces,20-50μm, surrounded by pale brown entostromata of texura globose or amorphous material. Locules globose,90-210×200-600μm diam, simple undivided, not sharingcommon walls, brown walls of textura epidermoidea. Conidiophores, frequently branchedabove the base near the mid-height, with up to four verticillate phialides,12-15×1-1.5μminclusive of phialides, embedded in a continuous gelatinous matrix. Conidiogenous cellssubcylindrical phialides, tapering to the apices, with minute collarettes and periclinalthickenings,9-17×0.7-1μm. Conidia hyaline, eguttulate, allantoid, aseptate,4.2-5.5×0.7-1.0μm.341representative isolates of C. chrysosperma confirmed by morphology and moleculardata were selected according to their obvious culture characters to study the genetic diversityusing ITS-rDNA sequence analysis and random amplified polymorphic deoxyribonucleic acid(RAPD) marker. Sequence analysis results showed that there were27different base in sequenceamplied from ITS-rDNA, the mean overal genetic distance was0.001, MP phylogenetic treecould not differentiate the strains which was clustered in one main clade. The RAPD resultdemonstrated all the random primers amplified reproducible polymorphic banding patterns. Theevaluation of genetic diversity got the79polymorphism bands, which polymorphic loci (p) wasof91.86%. Based on0.41Dice similarity coefficient, cluster analysis of the data divided theisolates into4groups which were almostly correspond to the groups subdivided by culturefeatures, showing a high genetic diversity among populations of C. chrysosperma. The resultalso demonstrated that there was no correlation between geographical origins and the resultinggroups of RAPD analysis. The amount of observed polymorphism showed by RAPD indicatedthat it was appreciate to study genetic diversity in C. chrysosperma, and could be used in otherspecies of Cytospora spp. and associated teleomorphs. |
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