3.3.20
IDENTIFICATION AND ANALYSIS OF PYTHIUM SPECIES IN SUGARCANE USING REAL TIME PCR DETECTION BY TAQMANTM

LA HEELAN1,2, BJ BASSAM1, BJ CROFT1, R DIETZGEN3 and DJ MACLEAN1,2

1Cooperative Research Centre for Tropical Plant Pathology, 2Department of Biochemistry, University of Queensland, and 3Queensland Agricultural Biotechnology Centre, St Lucia, Qld 4072, Australia

Background and objectives
Sugarcane is an agronomically important crop grown along the north-east coast of Australia. Pythium arrhenomanes and P. graminicola are oomycete phytopathogens that have been demonstrated to cause root disease of sugarcane in glasshouse tests [1]. They contribute to poor root syndrome which manifests as a poor root system and stunted growth of the plant. These two species are morphologically very similar and hence difficult to distinguish by traditional taxonomic identification. We have sequenced the complete ITS region of rRNA genes of representative isolates of these and other Pythium species, to assess phylogenetic relationships and to identify nucleic acid sequence differences, and hence develop PCR-based procedures for detection, identification and quantitation of these pathogens in root samples. We are currently developing TaqManTM-based tests that use real-time fluorescence measuring during PCR for qualitative and quantitative detection.

Materials and methods
The ITS region of 37 isolates representing Pythium arrhenomanes, Pythium graminicola and 15 other Pythium species were sequenced. Sequences were aligned with Clustal W and analysed using Phylogenetic Analysis Using Parsimony (PAUP). PCR primers homologous to ITS sequences were designed to span the 5.8s rRNA gene. Primers for gel-based detection amplify 343- and 708-bp amplicons of P. arrhenomanes and P. graminicola, respectively. These primers can be used in a multiplex PCR to detect one or both pathogens in a sample. Primers and probes for TaqManTM assays were designed according to criteria described in [1], and specific amplicons were detected using the ABI Prism model 7700 Sequence Detector (Perkin Elmer).

Results and conclusions
PAUP analysis of the Pythium ITS sequences indicated two divergent phylogenetic groups, with P. arrhenomanes and P. graminicola branching within the group that included P. myriotylum and P. dissotocum; the other group included P. irregulare and P. ultimum. P. arrhenomanes and P. graminicola display 20% sequence divergence from each other in the ITS regions. Gel-based detection was achieved with highly specific PCR primers that amplified 343- and 708-bp amplicons, respectively, for P. arrhenomanes and P. graminicola. TaqManTM assays have been developed for amplicons spanning the 5.8s rRNA gene within the ITS region of the rDNA tandem repeat, using hybridization probes homologous to the 5.8s gene. These amplicons are longer than the 50-150-bp sequence recommended by [1], and will be compared with other options for primer/probe design. Assessment of sensitivity, amplification efficiency and suitability for quantitative assays will be discussed.

References
1. Lee YS, Hoy JW, 1992. Plant Disease 76, 735-739.
2. Bassam BJ, Allen T, Flood S et al., 1996. Australasian Biotechnology 6, 285-294.