Detection and Identification ofMonosporascusspp. with Genus-Specific PCR Primers and Nonradioactive Hybridization Probes

Abstract

Methodology was developed and evaluated for purposes of polymerase chain reaction (PCR)-mediated detection of Monosporascus cannonballus, a soilborne ascomycete causing root rot/vine decline on Cucurbitaceae. In previous studies the sequence of the internal transcribed spacer (ITS) region of the ribosomal DNA unit was shown to be conserved within the genus Monosporascus yet different from that of fungi taxonomically and ecologically most closely related. Among five PCR primers derived from the ITS region a primer pair was selected that amplified the DNA from infected roots most efficiently and most consistently. The method developed for DNA extraction and included as part of the PCR-detection protocol uses 10-mg samples, and requires neither incubation nor organic solvents. The use and applicability of the method is illustrated for fresh or dry roots, individual ascospores, and processed soil samples. The detection method is based on amplifying the DNA by PCR with Monosporascus-specific PCR primers (B. R. Lovic et al., 1995, Phytopathology 85 :655-661), performing agarose gel electrophoresis followed by dot blot hybridization with digoxigenin-labeled portions of the Monosporascus ITS region. The duration of the procedure and amounts and hazardous nature of the chemicals have been minimized for each step. The average duration of the procedure for 30 root samples, including DNA extraction, PCR amplification, and gel electrophoresis, is less than 7 h. This detection method proved especially valuable for identifying a relatively large (20%) portion of Monosporascus population that does not produce perithecia.