Abstract
The internal transcribed spacer regions of the ribosomal DNA repeat unit in representative isolates of Monosporascus cannonballus and M. eutypoides were amplified by PCR and sequenced. The PCR-amplified ITS regions of 13 isolates representing all geographic regions where Monosporascus spp. occurs were analyzed with nine restriction enzymes and determined to be identical. Five PCR primers were synthesized based on the DNA sequence of the ITS 1 and 2. PCR amplifications using these primers consistently yielded products of predicted sizes from the DNA of all isolates of Monosporascus spp. but not from the DNA of any other fungi tested. The identity of the amplification products was confirmed as Monosporascus spp. ITS by Southern hybridizations with a digoxigenin-labeled probe. The ITS primers and probes were further developed into a method for detecting Monosporascus spp. in muskmelon roots and soil. Fifteen isolates of Monosporascus spp. were tested for pathogenicity to muskmelon. While most isolates performed consistently, several caused a different level of reduction in plant growth parameters in replicated experiments. The variable isolates were also characterized by a degenerated morphology and by the presence of low-molecular-weight nucleic acid fragments. These fragments were determined to be dsRNA based on resistance to RNase under high salt conditions, green fluorescence following acridine-orange staining, and purification by CF11 chromatography. Individual fragments could be transferred between different isolates and such transfers were associated with a change of phenotype. Two commercial muskmelon fields were sampled for presence of M. cannonballus using a hierarchical sampling design. M. cannonballus was isolated from 88% of the plants. More than 70% of the isolates contained one to six dsRNA fragments that varied in size from 1.8 kb to 3.8 kb relative to a dsDNA size marker. Seven dsRNA length polymorphisms (profile) were found among 250 isolates. There was no evident spatial aggregation of the isolates characterized by the same dsRNA profile and up to five different dsRNA profiles were detected in the isolates originated from the same root.
Lovic, Branislav, R. (1994). Molecular studies of Monosporascus spp. : applications in diagnostics, taxonomy, population biology, and control. Texas A&M University. Texas A&M University. Libraries. Available electronically from
https : / /hdl .handle .net /1969 .1 /DISSERTATIONS -1554728.