2.2.48
KARYOTYPIC ANALYSES OF FIVE FUSARIUM SPP. CAUSING WHEAT SCAB BY FLUORESCENCE MICROSCOPY AND FLUORESCENCE IN SITU HYBRIDIZATION

T SATO1, M TAGA2, H SAITOH1, T NAKAYAMA1, T TAKEHARA1

1 Upland Crop Disease Laboratory, National Agriculture Research Center, Kannondai, Tsukuba, Ibaraki 305-8666, Japan: 2Department of Biology, Faculty of Science, Okayama University, Tsushima, Okayama 700-0082, Japan

Background and objectives
Wheat scab is caused by several Fusarium spp. and investigation of karyotypes is essential to understand the mechanisms of genetic variation in natural populations of these fungi. At present, we have little karyotypic information for them from either conventional meiotic cytology or pulsed field gel electrophoresis because they are frequently unamenable to these karyotyping methods. The objective of this study is to obtain karyotypic data for these fungi by using a rather new cytological method that combines the germ tube burst method (GTBM) and fluorescence microscopy. It also aims to correlate the karyotypic data with their taxonomy.

Materials and methods
Five Fusarium species, F. ;graminearum, F. ;culmorum, F. ;crookwellense, F. ;avenaceum and F. ;acuminatum were examined. For each species, two strains was used for karyotyping and one strain for fluorescence in situ hybridization (FISH). Cytological karyotypes were determined by observation on specimens of somatic metaphase chromosomes prepared by GTBM[1] using fluorescence microscopy. Electrophoretic karyotypes were analysed by CHEF apparatus. FISH using rDNA from Alternaria alternata as a probe was done following the method of Taga and Murata [2].

Results and conclusions
Cytological karyotyping showed that chromosome number (CN) for F. ;graminearum, F. ;culmorum and F. ;crookwellense was 4, and that all of these chromosomes were large in size. These results are contradictory to the previous reports of Punithalingam, in which CNs of 6 and 8 were proposed for F. ;graminearum and F. ;culmorum, respectively. F. ;acuminatum had four large plus an additional small chromosome. Conversely, the CNs for F. ;avenaceum were apparently different from other species; it had 8-10 small-sized chromosomes. The data from electrophoretic karyotyping for F. ;graminearum, F. ;culmorum and F. ;acuminatum supported the cytological karyotypes. Using FISH with rDNA as a probe a single hybridization signal was observed in a filament-like form at the end of a specific chromosome in all five species, which indicates that the nucleolar organizer region (NOR) is solitary in each genome. The karyotypic data in this study led to the conclusion that three species (F. ;graminearum, F. ;culmorum and F. ;crookwellense) in the same section Discolor share a similar karyotype and that the species belonging to the other sections (F. ;avenaceum in Roseum and F. ;acuminatum in Gibbosum) have distinctly different karyotypes.

References
1.Shirane N, Masuko M, Hayashi Y, 1988. Phytopathology 78, 1627-1630.
2.Taga M, Murata M, 1994. Chromosoma 103, 408-413.