2.2.34
GENOTYPIC DIVERSITY AMONG GLOBAL ISOLATES OF SUNFLOWER RUST REVEALED BY GENETIC MARKERS
BC ENGEL1, EAB AITKEN1, KC GOULTER1, TJ GULYA2, GA KONG 3 and JK KOCHMAN 3

1CRC for Tropical Plant Pathology, University of Queensland, St Lucia, Queensland 4072, Australia; 2United States Department of Agriculture, PO Box 5677 State University Station, Fargo, North Dakota 58105, USA ; 3Queensland Department of Primary Industries, PO Box 102, Toowoomba, Australia

Background and objectives
Rust disease caused by the fungus Puccinia helianthi Schw. is a serious constraint to the economic potential of sunflower crops in Australia and worldwide. The expansion of sunflower production in Australia in the late 1960s has resulted in a rapid, stepwise increase in virulence of sunflower rust on commercial hybrids. Similar trends have been noted in other sunflower cropping countries such as the the USA, Canada and South Africa. Because shifts in the virulence of P.  helianthi occur frequently, an understanding of the genotypic diversity in sunflower rust populations is essential for the effective deployment of rust resistance genes in sunflower. The aim of this investigation was to assess the extent of genotypic diversity of sunflower rust in Australia and worldwide using neutral genetic markers.

Materials and methods
DNA was extracted from 21 isolates of P. helianthi collected from commercial and wild sunflowers in Australia over the period 1978-1996. These isolates were increased from single uredinia and pathotyped. DNA was also extracted from 22 isolates maintained at Fargo, North Dakota, USA. These isolates included representatives of the major North American pathotypes (Races 1, 2, 3, 4 and 6) as well as uncharacterized isolates from Europe, Africa, South America and China. An assessment of genotypic diversity was made by comparing DNA fingerprints generated using RAPD-PCR. PCR amplification of the ITS region of the ribosomal gene repeat unit using primers targeted to conserved sequences [1], and PCR amplification of the IGS region with primers targeted to sequences within the Puccinia spp. 5S ribosomal gene [2] was also performed. Amplified products were resolved by polyacrylamide gel electrophoresis and detected by silver staining. Computer-aided data analysis of the DNA fingerprints was performed using Bio-Rad Molecular Analyst software [3].

Results and conclusions
Analysis of the DNA fingerprints generated with RAPD-PCR revealed a variable number of polymorphic bands among isolates. Australian isolates showed a higher level of genotypic diversity compared with global isolates. Among Australian isolates, several polymorphisms were detected even between different isolates of the same pathotype. These observations indicate that there may have been several introductions of P. helianthi into Australia and that mutation to the same virulence has occurred in a number of different genetic backgrounds. A preliminary phenetic analysis of DNA fingerprints supports this hypothesis since isolates of different pathotypes cluster together. Mutations to virulence in different genetic backgrounds have been selected in response to the different resistance genes deployed in commercial hybrids, allowing divergent pathotypes to proliferate. Sexual crosses have been established to determine whether genotypic and pathotypic variation can also be generated through the sexual cycle. Variation in the IGS and ITS regions of the ribosomal repeat unit of the rust isolates has been detected and differences between isolates are being investigated by sequencing. A more extensive analysis of DNA fingerprints, IGS and ITS is being undertaken to determine the phylogenetic relationships between Australian and global isolates of sunflower rust.

References
1. White TJ, Bruns T, Lee S, Taylor J, 1990. In: MA Innis MA et al., eds, PCR Protocols: A Guide to Methods and Applications. London: Academic Press.
2. Fox SL, Harder DE, Kim WK, 1995. Canadian Journal of Plant Pathology 17, 197-204.
3. Bentley S, Bassam BJ, 1996. Journal of Phytopathology 144, 207-13.