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ARS Home » Plains Area » Fargo, North Dakota » Edward T. Schafer Agricultural Research Center » Sugarbeet and Potato Research » Research » Publications at this Location » Publication #101860

Title: ACTIN CODING SEQUENCES AND RRNA GENE ITS REGIONS AS TARGETS FOR PCR-BASED DIFFERENTIATION AND DETECTION OF SUGARBEET FUNGAL PATHOGENS

Author
item Weiland, John
item Sundsbak, Jamie

Submitted to: Plant Disease
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/26/2000
Publication Date: N/A
Citation: N/A

Interpretive Summary: Fungal pathogens cause the most serious losses due to disease in sugarbeet in the U.S. These pathogens can be divided into those causing foliar diseases and those causing seedling or root rot diseases. Within these two groups, it often is difficult to positively identify which fungus is causing the disease. Molecular (DNA) fingerprinting has influenced the identification of organisms from humans, in forensic or criminal investigations, to agriculture, such as in the patenting of plant genetic resources. Here we show that DNA fingerprinting can be applied to the rapid identification of sugarbeet fungal pathogens, even before they are isolated from the diseased plant material. The information shows that ambiguity in the typing of the disease-causing organism can be reduced by using techniques described, which in turn can lead to more timely and appropriate disease-control measures.

Technical Abstract: The DNA sequences of the actin genes of several fungi were compared and highly conserved regions in the coding sequence were identified. Deoxyoligonucleotide primers were synthesized based on conserved sequence blocks in the 5' and 3' ends of the open reading frame encoding the actin protein. In addition, primers (ITS1 and ITS4) based on conserved regions of the ribosomal RNA (rRNA) genes of fungi were synthesized. Use of the primers in the polymerase chain reaction (PCR) resulted in the amplification of DNA products from the genomes of sugarbeet fungal pathogens of a size consistent with the amplification of the actin gene and rRNA gene sequences, respectively, in these fungi. With one primer pair (5FWDACT and MIDREVACT) directed to the actin gene , the major products amplified from the DNA of Aphanomyces cochlioides, Pythium ultimum, Cercospora beticola, Phoma betae, Fusarium oxysporum, and Rhizoctonia solani were of the sizes of 0.9, 0.9, 1.1, 1.1, 1.2 and 1.7 kilobasepairs (kbp), respectively, whereas no product was generated from the DNA of sugarbeet (Beta vulgaris L.). Restriction endonuclease digestion of products amplified using 5FWDACT and MIDREVACT permitted the differentiation of A. cochlioides from A. euteiches. Use of ITS1 and ITS4 in PCR reactions employing the same template DNAs and reaction conditions yielded single products of 0.7, 0.8, 0.5, 0.5, 0.6, and 0.7 kbp, respectively, as well as a 0.7 kbp product from DNA of sugarbeet. The data indicate that actin and rRNA gene sequences are appropriate targets for the development of PCR-based strategies for distinguishing sugarbeet fungal pathogens at the genus level. The presence of A. cochlioides DNA in extracts of diseased sugarbeet seedlings was detected