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Development of a DNA based diagnostic assay for the detection and differentiation of pathogenic fusarium oxysporum isolates from conifers in the Pacific Northwest Donaldson, Gary C.

Abstract

Fusarium oxysporum is commonly associated with a variety of diseases in bare-root conifer nurseries. The identification of F. oxysporum typically requires considerable expertise because of the morphological similarity of many Fusarium species. Furthermore, different isolates of F. oxysporum may or may not be pathogenic. Among pathogens, a large degree of variation in virulence occurs. There were two primary objectives of this work. The first was to continue the development of a previously described DNA-based diagnostic assay which relies on restriction fragment length polymorphisms (RFLPs) within the histone-H3 encoding gene to identify several Fusarium species, including F. oxysporum. The second was to expand the capabilities of the assay to differentiate pathogenic and nonpathogenic isolates of F. oxysporum, or at least opposite ends of the virulence spectrum. To confirm the utility of the DNA-based diagnostic, blind tests were performed using a variety of Fusarium species and fungi commonly isolated from nursery environments. Genetic diversity within tissue and soil subpopulations of F. oxysporum was examined at two nurseries in the state of Oregon to determine the degree of genetic differentiation among nursery populations. To screen for genetic markers associated with virulence, amplified fragment length polymorphisms (AFLPs) derived from 9 different primers were compared to the phenotype of 24 isolates of F. oxysporum selected for their high or low virulence. During the course of this work, evidence that suggests F. oxysporum is a complex of species has accumulated. Although this complicated the screening process, within the limited set of high and low virulence isolates studied, a large number of markers does allow separation of high and low virulence isolates. There is small, but significant, genetic differentiation of the Bend and Medford populations. The relatively low level of differentiation suggests that either the populations at the two sites originated from the same source, or contaminated material moves between the two sites. All of the available data suggest that F. oxysporum has a clonal population structure. Such a population is well suited to DNA-based diagnostic assays.

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