Selection and characterization of genomic DNA clones of Pyrenophora teres and their application for disease diagnosis via the polymerase chain reaction (PCR)

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Montana State University - Bozeman, College of Agriculture


Polymerase Chain Reaction (PCR) protocols were developed for the diagnosis of net and spot forms of Pvrenophora teres. Low copy number sequences selected from a P. teres f. sp. maculata random genomic library were used as a source of probes. Emphasis was placed on those sequences identifying DNA polymorphisms between net and spot isolates and with little or no sequence similarity with barley, wheat, or triticale genomes. Sequences identifying a large deletion in genomic DNAs of net and spot isolates were preferred over sequences detecting small DNA changes. Sequence data of two informative clones, pPtm-290, and pPtm-60, were used to construct primer sets to amplify the corresponding sequence in genomic DNAs of net and spot isolates present in barley plants infected with these pathogens. PCR results demonstrated the potential of the PCR as a diagnostic tool for P. teres. All the PCR experiments conducted with primers designated as Pt-1 and Pt-2 constructed using the sequence data from pPtm-290, showed a strict correlation between the presence of a 430 bp band and the presence of the pathogen in genomic DNAs of barley infected with the net form, spot form or both pathogens. PCR experiments with primers Pt-3 and Pt-4 constructed using sequence data from pPtm-60, indicated that it is possible to detect polymorphic bands between net and spot isolates as evidenced by the PCR products analyzed in an ethidium bromide agarose gel. PCR analysis offers a sensitive, rapid, inexpensive, and non-radioactive technique for the diagnosis of P. teres infection in field-grown barley plants. Future experiments should focus on the ability of the PCR to detect P. teres and P. graminea in infected barley seeds. Additionally, PCR-based protocols for P. teres diagnosis could possibly be incorporated in seed certification programs to avoid the distribution of infected seed in farmer fields.




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