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Molecular Plant Pathology

Sequence Analysis of the ITS Regions of rDNA in Monosporascus spp. to Evaluate Its Potential for PCR-Mediated Detection. B. R. Lovic, Graduate research assistant, Department of Plant Pathology and Microbiology, Texas A&M University, College Station 77843; R. D. Martyn(2), and M. E. Miller(3). (2)Professor, Department of Plant Pathology and Microbiology, Texas A&M University, College Station 77843; (3)Associate professor, TAMU, AREC, Weslaco 78596. Phytopathology 85:655-661. Accepted for publication 1 March 1995. Copyright 1995 The American Phytopathological Society. DOI: 10.1094/Phyto-85-655.

Monosporascus cannonballus and M. eutypoides are recently described soilborne ascomycetes that cause root rot/vine decline of cucurbits. The internal transcribed spacer (ITS) regions 1 and 2 of the ribosomal DNA (rDNA) from the representative isolates of the two species were amplified by polymerase chain reaction (PCR) using conserved ITS primers from conserved regions of the 18S, 5.8S, and 28S ribosomal genes. The size of the entire spacer region, including the 5.8S gene, was estimated to be 610 bp based on gel electrophoresis of the PCR-amplified product. The entire region was cloned into a pUC18 vector, sequenced, and the DNA sequence aligned with published sequences from other fungi to identify gene-spacer junctions. Comparison to the published sequences of other fungi revealed up to 90% homology at the 3’ end of the 18S gene, up to 79% homology with the 5.8S gene and up to 74% homology at the 5’ end of the 28S gene. The DNA sequences (ITS 1 and ITS 2) contained within these regions did not show homology with any published DNA sequences. The ITS regions from 12 isolates of Monosporascus spp. representing all geographic regions of their reported occurrence were PCR-amplified and digested singularly with nine restriction enzymes but no length polymorphisms could be detected. The sequences of the ITS 1 and ITS 2 were used to construct five PCR primers, each of which was subsequently shown to amplify identical, predicted-size, fragments from the DNA of all 12 Monosporascus isolates tested. PCR primers from the ITS regions of Monosporascus spp. failed to amplify predicted-size fragments from the DNA of any other soil-borne fungi tested including representative species of the genera considered taxonomically (e.g., Anixiella) and ecologically (e.g., Fusarium., Stagonospora, Macrophomina, etc.) most closely related to Monosporascus spp. These primers were also tested on the DNA extracted from the roots of Monosporascus-infected muskmelon and were shown to consistently amplify the predicted-size fragments whose identity was further confirmed by Southern hybridization to the digoxigenin-labeled portions of the ITS regions. In addition to establishing that the DNA sequence of the ITS region is conserved within the genus Monosporascus this study also illustrates a convenient approach to developing “molecular tools” for detection of plant pathogenic fungi without necessarily having any detailed knowledge of their genome organization.

Additional keywords: PCR diagnostics.