Rallos, Lynn Esther E.2014-07-172014-07-172013-01-21vt_gsexam:248http://hdl.handle.net/10919/49594Practical resistance of Erysiphe necator to quinone outside inhibitors (QoIs) is now widespread, and resistance to demethylation inhibitors (DMIs) has also developed.  The goal of this research was to characterize fungicide resistance by elucidating resistance mechanisms and determining its stability.  QoI resistance persisted for several years in a field population after QoI application ended.  Resistant isolates were highly competitive in mixed populations in competition assays under laboratory conditions, indicating a lack of fitness cost.  In one competition trial under field conditions, resistance frequency declined, possibly due to spore migration and influx of background inoculum, but in a second trial, it did not decline.  Double resistance to QoI and DMI was detected and DMI application may have been partially responsible for maintaining QoI resistance in the field.  One isolate with QoI resistance but an undetectable level of the major QoI mutation was shown to be heteroplasmic -- resistant strains could be selected from this isolate. DMI resistance mechanisms in E. necator included the Y136F mutation in CYP51 and cyp51 over-expression.  The first mechanism was present in almost all isolates with substantial levels of resistance, and cyp51 expression level was correlated with resistance level.  Three cyp51 genotypes were detected.  Wildtype isolates with the TAT genotype were sensitive to DMIs, while isolates with increased resistance had either a TTT or TWT genotype; TWT indicated the presence of both wildtype and mutant alleles.  Cyp51 was expressed 1.4 to 19 times more in mutants than in wildtype.  It is not known whether the significant differences in cyp51 expression level among isolates and among genotype groups are due to gene copy number variation.  DMI resistance was found to decline after years of subculturing, and the decline appeared to occur after a few culture transfers of field samples on fungicide-free host leaves.  The observed decline, together with the finding that isolates could be "trained" to increase resistance, and may be slightly induced in cyp51 expression when successively challenged to grow in increasing fungicide concentration, indicate instability of DMI resistance.ETDIn CopyrightErysphe necatorfungicide resistanceQoIDMIcytochrome bCYP51resistance mechanismsDissertation