A Quantitative Assay of Puccinia coronata f. sp. avenae DNA in Avena sativa.

Crown rust, caused by Puccinia coronata f. sp. avenae, is the most damaging disease of oat. Quantification of the disease can be done by visual or digital assessments of diseased leaf area, lesion number, lesion size, and latent period. Laborious measurements of sporulation can also be made. As an alternative to these methods, a new quantitative assay was developed. The method employs simple inoculum application, quantitative sampling from inoculated areas, a closed tube DNA extraction method restricting loss of tissue, and real-time polymerase chain reaction (PCR) using a pathogen-specific TaqMan primers/probe set. Image analyses of genotypes with varying levels of crown rust resistance were compared to fungal DNA (FDNA) estimations by the new assay. The moderately resistant genotype IA98822-2 was distinguished from susceptible genotypes at the seedling stage, and the moderately resistant genotype O × T 107 was distinguished from the resistant cultivar TAM-O-301 at seedling and adult plant stages using FDNA. These separations were not evident with digital image analysis. The new assay also detected fungal development earlier and more rapidly in genotypes with lower levels of resistance. The assay can consistently estimate disease and should be useful for studying many aspects of the crown rust host-pathogen interaction where precise assessment of pathogen development is needed.

[1]  J. A. Bates,et al.  The application of real-time PCR to the identification, detection and quantification of Pyrenophora species in barley seed. , 2001, Molecular plant pathology.

[2]  J. Parlevliet.  Resistance of the non-race-specific type , 1985 .

[3]  C. Osborne,et al.  A comparison of visual and digital image-processing methods in quantifying the severity of coffee leaf rust (Hemileia vastatrix) , 1993 .

[4]  E. Milus,et al.  Characterization of resistance to leaf rust in Pacific Northwest wheats. , 1980 .

[5]  A. Heagle,et al.  Some effects of moderate adult resistance to crown rust of oats. , 1970 .

[6]  H. Judelson,et al.  Enhanced Polymerase Chain Reaction Methods for Detecting and Quantifying Phytophthora infestans in Plants. , 2000, Phytopathology.

[7]  G. Hartman,et al.  Detection and Quantification of Fusarium solani f. sp. glycines in Soybean Roots with Real-Time Quantitative Polymerase Chain Reaction. , 2004, Plant disease.

[8]  R. F. Peterson,et al.  A DIAGRAMMATIC SCALE FOR ESTIMATING RUST INTENSITY ON LEAVES AND STEMS OF CEREALS , 1948 .

[9]  C. Lannou,et al.  Wheat Leaf Rust Uredospore Production on Adult Plants: Influence of Leaf Nitrogen Content and Septoria tritici Blotch. , 2004, Phytopathology.

[10]  A. Vecchione,et al.  Development of a High-Throughput Method for Quantification of Plasmopara viticola DNA in Grapevine Leaves by Means of Quantitative Real-Time Polymerase Chain Reaction. , 2005, Phytopathology.

[11]  J. Parlevliet.  Pleiotropic association of infection frequency and latent period of two barley cultivars partially resistant to barley leaf rust , 1986, Euphytica.

[12]  H. Ohm.  Three Components of Slow Leaf-Rusting at Different Growth Stages in Wheat , 1976 .

[13]  K. Politowski.  Effect of Temperature, Light, and Dew Duration on Relative Numbers of Infection Structures of Puccinia coronata avenae , 1975 .

[14]  Yinong Yang,et al.  Quantification of Magnaporthe grisea During Infection of Rice Plants Using Real-Time Polymerase Chain Reaction and Northern Blot/Phosphoimaging Analyses. , 2002, Phytopathology.

[15]  J. E. Diaz-Lago,et al.  Evaluation of Components of Partial Resistance to Oat Crown Rust Using Digital Image Analysis. , 2003, Plant disease.

[16]  Evaluation of components of resistance in oat breeding lines and cultivars to crown rust (Puccinia coronata f. sp. avenae) under controlled environmental conditions , 1995 .

[17]  J. Holland,et al.  A linkage map of hexaploid oat based on grass anchor DNA clones and its relationship to other oat maps. , 2001, Genome.

[18]  Forrest W. Nutter,et al.  Assessing the accuracy, intra-rater repeatability, and inter-rater reliability of disease assessment systems , 1993 .

[19]  K. Livak,et al.  Real time quantitative PCR. , 1996, Genome research.

[20]  J M Henson,et al.  The polymerase chain reaction and plant disease diagnosis. , 1993, Annual review of phytopathology.

[21]  R. D. Wilcoxson,et al.  A device for uniform deposition of liquid-suspended urediospores on seedling and adult cereal plants , 1984 .

[22]  A. Roelfs.  Genetic Control of Phenotypes in Wheat Stem Rust , 1988 .