Survey and Genetic Analysis of Demethylation Inhibitor Fungicide Resistance in Monilinia fructicola From Michigan Orchards.

Resistance to sterol demethylation inhibitor (DMI) fungicides in Monilinia fructicola, causal agent of brown rot of stone fruit, has been reported in the southeastern and eastern United States and in Brazil. DMI resistance of some M. fructicola isolates, in particular those recovered from the southeastern United States, is associated with a sequence element termed "Mona" that causes overexpression of the cytochrome demethylase target gene MfCYP51. In this study, we conducted statewide surveys of Michigan stone fruit orchards from 2009 to 2011 and in 2019, and we determined the sensitivity to propiconazole of a total of 813 isolates of M. fructicola. A total of 80.7% of Michigan isolates were characterized as resistant to propiconazole by relative growth assays, but the Mona insert was not uniformly detected and was present in some isolates that were not characterized as DMI resistant. Gene expression assays indicated that elevated expression of MfCYP51 was only weakly correlated with DMI resistance in M. fructicola isolates from Michigan, and there was no obvious correlation between the presence of the Mona element and elevated expression of MfCYP51. However, sequence analysis of MfCYP51 from 25 DMI-resistant isolates did not reveal any point mutations that could be correlated with resistance. Amplification and sequencing upstream of MfCYP51 resulted in detection of DNA insertions in a wide range of isolates typed by DMI phenotype and the presence of Mona or other unique sequences. The function of these unique sequences or their presence upstream of MfCYP51 cannot be correlated to a DMI-resistant genotype at this time. Our results indicate that DMI resistance was established in Michigan populations of M. fructicola by 2009 to 2011, and that relative resistance levels have continued to increase to the point that practical resistance is present in most orchards. In addition, the presence of the Mona insert is not a marker for identifying DMI-resistant isolates of M. fructicola in Michigan.

Occurrence of QoI Resistance and Detection of the G143A Mutation in Michigan Populations of Venturia inaequalis.

Control strategies for Venturia inaequalis rely heavily on chemical fungicides. Single-site fungicides such as the quinone-outside inhibitors (QoI) have been used in Michigan apple orchards for more than 11 years. In 2008, we sampled eight commercial orchards in the Fruit Ridge growing region of Michigan in which apple scab control failures were observed on 'McIntosh' apple following applications of kresoxim-methyl or trifloxystrobin. QoI resistance was assessed in 210 total isolates (a total of 17 orchards) using a spore germination assay and in 319 isolates using a polymerase chain reaction (PCR) assay to detect the G143A mutation located within the V. inaequalis cytochrome b gene (CYTB). The G143A mutation is known to confer high-level QoI resistance in plant-pathogenic fungi. QoI resistance was confirmed in 50 and 64% of the isolates tested with the spore germination and PCR assays, respectively, and there was a 97% concordance observed between the assays. In 2009, we sampled and examined an additional 1,201 V. inaequalis isolates from 64 orchards in Michigan and 86 isolates from four baseline sites in Ohio. All of these isolates were assayed for the G143A mutation and it was detected within 67 and 0% of the Michigan and Ohio isolates, respectively. Our results indicate the widespread occurrence of QoI resistance in Michigan commercial orchard populations of V. inaequalis. Loss of QoI fungicides further limits the arsenal of fungicides available to commercial apple growers for successful scab management.