Germination Of Teliospore Of Tilletia Controversa Kǖhn And Polymorphism Of Its Ribosomal DNA

Dwarf bunt of wheat, caused by Tilletia controversa Kuhn (TCK), is a destructivedisease on wheat, as well as an important international quarantine disease. Since 1970, it hasbeen an important issue in China- America wheat trading. However, the morphologicalcharacteristic of TCK is similar with T. foetida Wallr. (TFL), T. caries Tul. (TCT) severalother related species. The traditional methods for diagnosis and detection of the disease weremostly based on the morphological characteristics, germination test of teliospores, and thesymptom of disease in the field, resulting in a long procedure and low accuracy. Therefore, itis imperative to develop a molecular assay for rapid identification and accurate detection ofTCK.The teliospore of Tilletia controversa kuhn could germinate on water agar, soil-extractagar and T19 agar. Soil-extract agar was the optimum media for germination of TCKtelispores and the concentration of teliospore suspension was approximately 500,000 permillilitre. It is about 20 days, since cultured, that the teliospores begin to germinate and 35～40 days latercould be transferred to the liquid media if large quantity mycelium was needed. The teliosporesuspension with the different concentration of NaClO displayed different germinationpercentage. The highest germination percentage in all of six treatments was obtained underthe 0.3MNaClO in the teliospore suspension for 30 minutes. The germination peak wasbetween 35 and 50 days. The mycelium could grow in PDA and T19 culture media. But T19liquid media was suitable for high production of mycelium. D-Biotin could enhance thegermination percentage of teliospore, while Nicotinic Acid,ρ-aminobenzoic acid and D—Pantothenic showed just inhibition effect. The fungi utilized a few of Adenine, Uracil andCytosine.Ten geographical isolates of Tilletia controversa Kuhn and several related species werecollected from the United States and China, respectively. Genomic DNA of fungal teliospores(10mg or so) was extracted after high moisture keeping in mortars for 24 hours using amodified benzyl chlorine. The quality and quantity of genomic DNA extracted by this methodwere basically suitable for RAPD, RFLP, AFLP analysis and even other common molecularbiological experiments. The ITS region of ribosomal DNA was amplified by a pair of primersITS1/ITS4.The amplified ITS fragments were a 500～750bp band, The amplified ITS regionwas digested with restrition endonucleases EcoR I, Pst I, Mse I. This reaction production were separated by polyacrylamide gel electrophoresis. The result showed that a 250bpspecific band was gotten in the Tilletia controversa Kuhn isolates. This fragment was presentin all of ten geographical isolates of Tilletia controversa Kuhn, but was not present in theTilletia laevis Kuhn [syn. Tilletia foetida(Wallr.) Liro], Tilletia caries(DC)Tul [syn.Tilletiatirtici(Bjerk.) Wint], Ustilago tritici Schaff and Sphacelotheca reiliana (Kuhn) Clint. Thespecific DNA fragment was isolated, cloned and sequenced, then,anticipatively, a sequencecharacterized amplified regions (SCAR)would be gotten through the conversion of RFLPmaker with primer designing and selection.