Content area
Abstract
A pair of PCR primers, NMS1 and NMS2, that amplify a mitochondrial small rRNA gene region of ascomycetes were designed. RFLP analysis of the amplified region differentiated eight fungal genera and DNA sequence analysis differentiated 10 Verticillium species. NMS1 and NMS2 thus may be used for identifying DNA probes at the below genus level and as mitochondrial markers for studying fungal cytoplasmic inheritance. Based on the sequence information of this region from V. dahliae, V. albo-atrum and V. tricorpus, PCR primers VMS1 and VMS2 were designed and found to be V. dahliae specific.
A rapid and versatile approach to generating specific DNA probes using RAPD was devised. This approach does not require a priori DNA sequence information of the organisms. Using this approach, a 567 bp DNA fragment was identified and found to exist only in V. dahliae. There was no sequence homology between this locus and any other known sequence. PCR primers VDS1 and VDS2 from this region were found to be specific to V. dahliae. An internal control for competitive PCR was constructed using a method that does not require additional PCR primers or target sequence information. The V. dahliae-specific PCR primers and the internal control aided in more rapid and specific detection of the pathogen directly in plant samples.
Using RAPD analysis, the genetic relationship of 34 isolates of V. dahliae, representing four Vegetative Compatibility Groups (VCGs) based on nit mutant analysis, was examined. It was found that the present VCG categorization only partially reflected the population structure of V. dahliae but further research is needed. The isolates originally assigned to VCG1 formed one group, designated RAPD Group I; isolates belonging to VCGs 2 and 4 formed RAPD Group II; and isolates in VCG2 and 3 formed RAPD Group III. All isolates from potato fell into RAPD Group II. All non-VCG 1 (RAPD Group I) cotton isolates belonged to RAPD Group III. Southern hybridization showed that co-migrating RAPD bands were homologous. A unique RAPD fragment that could serve as a VCG 1-specific probe was also identified.
