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GrainGenes Reference Report: PDS-85-220

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Reference	PDS-85-220
Title	A PCR-based assay to detect Rhynchosporium secalis, in barley seed
Journal	Plant Disease
Year	2001
Volume	85
Pages	220-225
Author	Lee HK Tewari JP Turkington TK
Abstract	Summary: A polymerase chain reaction (PCR)-based diagnostic assay was developed to detect Rhynchosporium secalis, the barley scald fungus, in barley seed. Species-specific primers were designed based on sequence data of a region consisting of the 5.8S RNA gene and internal transcribed spacers 1 and 2 of R. secalis. The sequenced regions showed 100% homology between the two R. secalis isolates and 93% homology between R. secalis and R. orthosporum. Five sets of synthesized oligonucleotide primers were tested for their specificity using 29 isolates of R. secalis of diverse geographic origins and from different barley cultivars. In addition, DNA extracts from 22 species of microbes either taxonomically related to or from the same niche as R. secalis were tested as negative controls. Among five sets of primers, a primer set, RS8 and RS9, was selected for use in detecting R. secalis because it amplified a 264-bp fragment from the DNA of all R. secalis isolates but not the DNA from other species used for validation of the specificity of this primer set. This primer set was also used to detect R. secalis in barley seed and successfully amplified the predicted size of the DNA fragment in the infected material. PCR detection of as little as 1 to 10 pg of R. secalis DNA was possible. The method described here requires 1 day for completion, compared to 10 days required for the cultural method
Keyword	[ Hide all but 1 of 29 ] assay barley barley cultivars barley scald chain detection dna fungus gene homologies homology hybridization isolate method monosporascus origin pcr poae polymerase polymerase chain reaction primer rhynchosporium rhynchosporium secalis scald seed sequence sequence data spacer specificity

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