Differentiation and Detection of Sugar Beet Fungal Pathogens Using PCR Amplification of Actin Coding Sequences and the ITS Region of the rRNA Gene

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 (internal transcribed spacer [ITS] regions 1 and 4) 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 sugar beet 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 kilobase pairs (kbp), respectively, whereas no product was generated from the DNA of sugar beet (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 sugar beet. The data indicate that actin and rRNA gene sequences are appropriate targets for the development of PCR-based strategies for distinguishing sugar beet fungal pathogens at the genus level. The presence of A. cochlioides DNA in extracts of diseased sugar beet seedlings was detected using PCR with primers 5FWDACT and MIDRE-VACT.