Submitted to: Journal of Sugarbeet Research
Publication Type: Abstract Only
Publication Acceptance Date: 1/11/1999
Publication Date: N/A
Technical Abstract: Determination of the causal agents of seedling damping-off and adult root rot can be confounded by the misclassification of the disease- causing organism and by the presence of co-colonizing saprophytes. The actin and nuclear ribosomal RNA (rRNA) genes were used as targets in the development of polymerase chain reaction (PCR) protocols that permitted discrimination of common sugarbeet fungal pathogens without the need for phytopathological expertise. Using DNA primers (5FWDACT and MIDREVACT) directed to conserved regions in the actin gene, amplified DNA was generated from genomic DNA prepared from Aphanomyces cochlioides, Pythium ultimum, Phizoctonia solani, Fusarium oxysporium, Phoma betae, and Cercospora beticola that was of a size consistent with the amplification of actin gene sequences. Use of primers ITS1 and ITS4 in the amplification of the internal transcribed spacer (ITS) region of the nuclear rRNA gene of these fungi also yielded products consistent with the amplification of this gene region. Size polymorphisms in the DNA amplified with the actin and rRNA primer pairs observed between pathogens in different genera also were consistent with the known sequence diversity that exists within these two genes. Where amplified product DNA size was indistinguishable between any two members of differing fungal genera, restriction fragment length polymorphisms (RFLPs) observed after restriction endonuclese digestion of the amplified DNA permitted discrimination of the pathogens. Use of the assay in the detection of A. cochlioides in infected sugarbeet seedlings is presented.