Title: An improved method to quantify Puccinia coronata f. sp. avenae DNA in the host Avena sativa. Authors
Submitted to: Canadian Journal of Plant Pathology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: January 1, 2010
Publication Date: June 2, 2010
Repository URL: http://riley.nal.usda.gov/nal_web/digi/submission.html
Citation: Acevedo, M. , Jackson, E. W. , Sturbaum, A. , Ohm, H. W. and Bonman, J. M.(2010) 'An improved method to quantify Puccinia coronata f. sp. avenae DNA in the host Avena sativa', Canadian Journal of Plant Pathology, 32(2):215-224. Interpretive Summary: Crown rust is the most important disease of cultivated oat causing significant yield loss and reduced seed quality. Host resistance is an effective and economical method of managing this important problem and to aid the development of resistant oat cultivars, we previously developed a precise method of assessing host resistance by measuring the amount of pathogen DNA within the host.. We have now improved the assay to make it much easier and less time consuming to use. The new assay can be used to improve our knowledge of how disease resistance is inherited which will in turn help scientist produce new resistant oat cultivars.
Technical Abstract: Identification and mapping of resistance loci against polycyclic pathogens such as rust can be enhanced by accurate measurement of various disease resistance components. In this study we successfully converted an absolute quantification assay of Puccinia coronata DNA to a relative assay by the adding a TaqMan® primers/probe set specific to the oat ß –actin gene. The new multiplex assay estimates the amount of fungal DNA in a sample relative to the amount of host DNA. The relative fungal DNA assay (RFDNA) developed reliably detected and quantified both host and pathogen DNA over five orders of magnitude. Using 12 oat cultivars that differ in their response to P. coronata isolate LGCG in repeated greenhouse experiments, the RFDNA assay detected differences equal to or greater than those detected with either digital image analysis of diseased leaf area (DLA) or absolute estimation of fungal DNA (AFDNA). Measuring crown rust resistance to LGCG using DLA, AFDNA and RFDNA in a P8669/P94163 recombinant inbred line population produced segregation ratios that did not differ from the 1:1 Medelian ratio expected for single gene. Compared to the FDNA assessment method, the RFDNA assay is faster and much easier to use yet has the same advantages of the FDNA method for precise quantification of the crown rust pathogen in oat leaves.