Submitted to: American Phytopathological Society Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: 5/20/2004
Publication Date: 7/20/2004
Citation: Okubara, P.A., Paulitz, T.C. 2004. Simultaneous identification and quantification of rhizoctonia solani and r. oryzae from root samples using real-time pcr. American Phytopathological Society Annual Meeting. Phytopathology 94: S77.
Technical Abstract: In the Pacific Northwest (PNW), Rhizoctonia damping off and root rot of small grain cereals are attributed to Rhizoctonia solani AG-8 and R. oryzae. The former is also associated with bare patch disease of wheat. Nucleotide sequence comparisons of the internal transcribed spacer (ITS) regions of Rhizoctonia indicate that PNW populations of R. oryzae and R. solani are comprised of at least three and four groups, respectively. PNW R. solani isolates, originally thought be of anastomosis group (AG) 8, shared significant ITS sequence identity to members of AG-2-1 and AG-10 in Genbank searches. AG has been correlated with ITS sequence for R. solani associated with bare batch of sugar beets in France. Our data indicate that PNW isolates associated with root rot and bare patch are more diverse than originally thought. We have developed an approach for designing real-time (quantitative) PCR primers that are specific to the ITS DNA of each lineage. The approach involves extensive scrutiny of primer and target sequences for potential PCR artifacts using a battery of sequence analysis software, and optimization of amplification conditions using a 96-sample Q-PCR thermocycler. A high throughput method for extraction of Rhizoctonia DNA from pure culture and plant material is being developed. Results of AG testing and primer performance will also be discussed. Our strategy for primer design and analysis can be applied to ITS sequences from any fungal pathogen, and can be extended to DNA sequences from bacterial and viral pathogens.