Submitted to: American Society of Sugarbeet Technologists
Publication Type: Proceedings
Publication Acceptance Date: 4/26/1999
Publication Date: N/A
Interpretive Summary: Several diseases in sugarbeet are caused by fungi affecting both the leaves and the root. Effective control of sugarbeet fungal pathogens requires that the fungus causing the disease be properly identified, since the chemicals to control one fungus may not be effective against another fungus. Typically, fungal identification can take from one day to several weeks to complete. The results presented here show that the major fungal pathogens of sugarbeet can be identified within a single day using DNA amplification technology. Adaptation of the protocols used in this basic research study may be of use in standard plant diagnostic labs. The rapid identification of pathogens affecting a crop will lead to more rapid implementation of control measures, thereby reducing crop losses for the producer. The data further will be of use to fungal taxonomists in efforts to place fungal groups on the evolutionary tree.
Technical Abstract: Determination of the causal agents of seedling damping-off and adult root rot can be confounded by 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, Rhizoctonia 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 endonuclease 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.