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Molecular and genetic studies of Fusarium moniliforme
Abstract
Fusarium moniliforme, a filamentous Ascomycete, is a complex species, consisting of six different mating populations. Fungal isolates within this species are distributed worldwide on many economically important plants. As part of our long term goal of the research to study fate, stability, and movement of introduced DNA into this fungus, three independent studies were conducted. Investigation of field strains revealed that distinct mating populations have differential sensitivity to the fungicide benomyl. The relatively high levels of native resistance to benomyl indicate that caution must be taken so that strains with engineered benomyl resistance can be distinguished from those with natural tolerance to benomyl. To examine the fate of introduced DNA at the chromosomal level, an electrophoretic karyotype was developed using pulsed-field gel electrophoresis. Ten chromosomal DNA bands were resolved on a CHEF gel with two migrating as doublets. Southern blot analysis of the CHEF gels using labeled telomere DNA and cDNA prepared from total mRNA of the fungus suggests that separated DNA molecules represent intact chromosomes and there are twelve chromosomes present in F. moniliforme. Cloned genes and random DNA sequences have been assigned to individual chromosomes. Examination of five strains from A mating population showed stable karyotypes regardless of geographical location and host preference. To further extend the stability studies, an altered $\beta$-tubulin gene was isolated, cloned, and sequenced from a UV irradiated benomyl-resistant mutant of F. moniliforme with the intent of using as a homologous marker. Molecular analysis of cloned $\beta$-tubulin gene revealed a number of properties characteristic of $\beta$-tubulin genes from other filamentous Ascomycetes. Results derived from Southern hybridization under low stringency suggested that two divergent $\beta$-tubulin genes exist in this fungus. The gene showed differential expression in different growth phases. In addition, a point mutation, which likely confers benomyl resistance, has been identified in the cloned $\beta$-tubulin gene, resulting in single amino acid substitution of asparagine for tyrosine at codon 50. The generated benomyl-resistant mutant had a cold-sensitive phenotype.
Subject Area
Genetics|Molecular biology|Plant pathology
Recommended Citation
Yan, Keying, "Molecular and genetic studies of Fusarium moniliforme" (1995). ETD collection for University of Nebraska-Lincoln. AAI9538660.
https://digitalcommons.unl.edu/dissertations/AAI9538660