Resistance to Boscalid, Fluopyram and Fluxapyroxad in Blumeriella jaapii from Michigan (U.S.A.): Molecular Characterization and Assessment of Practical Resistance in Commercial Cherry Orchards.

Management of cherry leaf spot disease, caused by the fungus Blumeriella jaapii, with succinate dehydrogenase inhibitor (SDHI) fungicides has been ongoing in Michigan tart cherry orchards for the past 17 years. After boscalid-resistant B. jaapii were first isolated from commercial orchards in 2010, premixes of SDHI fungicides fluopyram or fluxapyroxad with a quinone outside inhibitor were registered in 2012. Here, we report widespread resistance to fluopyram (FluoR), fluxapyroxad (FluxR), and boscalid (BoscR) in commercial orchard populations of B. jaapii in Michigan from surveys conducted between 2016 and 2019. A total of 26% of 1610 isolates from the 2016-2017 surveys exhibited the fully-resistant BoscR FluoR FluxR phenotype and only 7% were sensitive to all three SDHIs. Practical resistance to fluopyram and fluxapyroxad was detected in 29 of 35 and 14 of 35 commercial tart cherry orchards, respectively, in surveys conducted in 2018 and 2019. Sequencing of the SdhB, SdhC, and SdhD target genes from 22 isolates with varying resistance phenotypes showed that BoscS FluoR FluxS isolates harbored either an I262V substitution in SdhB or an S84L substitution in SdhC. BoscR FluoR FluxR isolates harbored an N86S substitution in SdhC, or contained the N86S substitution with the additional I262V substitution in SdhB. One BoscR FluoR FluxR isolate contained both the I262V substitution in SdhB and the S84L substitution in SdhC. These mutational analyses suggest that BoscR FluoR FluxR isolates evolved from fully sensitive BoscS, FluoS, FluxS isolates in the population and not from boscalid-resistant isolates that were prevalent in the 2010-2012 time period.

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.

A Method for the Examination of SDHI Fungicide Resistance Mechanisms in Phytopathogenic Fungi Using a Heterologous Expression System in Sclerotinia sclerotiorum.

Succinate dehydrogenase inhibitors (SDHIs) are a class of broad-spectrum fungicides used for management of diseases caused by phytopathogenic fungi. In many cases, reduced sensitivity to SDHI fungicides has been correlated with point mutations in the SdhB and SdhC target genes that encode components of the succinate dehydrogenase complex. However, the genetic basis of SDHI fungicide resistance mechanisms has been functionally characterized in very few fungi. Sclerotinia sclerotiorum is a fast-growing and SDHI fungicide-sensitive phytopathogenic fungus that can be conveniently transformed. Given the high amino acid sequence similarity and putative structural similarity of SDHI protein target sites between S. sclerotiorum and other common phytopathogenic ascomycete fungi, we developed an in vitro heterologous expression system that used S. sclerotiorum as a reporter strain. With this system, we were able to demonstrate the function of mutant SdhB or SdhC alleles from several ascomycete fungi in conferring resistance to multiple SDHI fungicides. In total, we successfully validated the function of Sdh alleles that had been previously identified in field isolates of Botrytis cinerea, Blumeriella jaapii, and Clarireedia jacksonii (formerly S. homoeocarpa) in conferring resistance to boscalid, fluopyram, or fluxapyroxad and used site-directed mutagenesis to construct and phenotype a mutant allele that is not yet known to exist in Monilinia fructicola populations. We also examined the functions of these alleles in conferring cross-resistance to more recently introduced SDHIs including inpyrfluxam, pydiflumetofen, and pyraziflumid. The approach developed in this study can be widely applied to interrogate SDHI fungicide resistance mechanisms in other phytopathogenic ascomycetes.

Draft Genome Sequence Resource for Blumeriella jaapii, the Cherry Leaf Spot Pathogen.

Blumeriella jaapii is the causal agent of cherry leaf spot (CLS), the most important disease of tart cherry in the Midwestern United States. Infection of leaves by B. jaapii leads to premature defoliation, which places trees at heightened risk of winter injury and death. Current management of CLS relies primarily on the application of three important fungicide classes, quinone outside inhibitors, sterol demethylation inhibitors, and succinate dehydrogenase inhibitors. Here, we present the first high-quality genome of B. jaapii through a hybrid assembly of PacBio long reads and Illumina short reads. The assembled draft genome of B. jaapii is 47.4 Mb and consists of 95 contigs with a N50 value of 1.5 Mb. The genomic information of B. jaapii, representing the most complete sequenced genome of the family Dermateaceae (Ascomycota) to date, provides a valuable resource for identifying fungicide resistance mechanisms of this pathogen and expands our knowledge of the phytopathogenic fungi in this family.

Boscalid Resistance in Blumeriella jaapii: Distribution, Effect on Field Efficacy, and Molecular Characterization.

Cherry leaf spot (CLS), caused by the fungus Blumeriella jaapii, is a major disease of tart cherry (Prunus cerasus L.) trees, leading to early defoliation that results in uneven ripening and poor fruit quality in the current season, reduced fruit set in the following season, and increased potential for winter injury and tree death. Pristine (BASF Corporation, Research Triangle Park, NC), a commonly used fungicide for CLS management in Michigan, is a premix of boscalid, a succinate dehydrogenase inhibitor, and pyraclostrobin, a quinone outside inhibitor. Reduced efficacy of Pristine for CLS control was observed in field trials and commercial orchards and highlights the importance of fungicide resistance monitoring. A total of 1,189 isolates from 31 commercial orchards in Michigan, 111 isolates from nontreated trees (four locations in Michigan and two locations in Ohio), and 133 isolates from a research orchard were collected during 2010, 2011, and 2012 and assayed on boscalid-amended media at concentrations ranging from 0 to 25 µg ml-1. Because of the very slow growth rate of B. jaapii in culture, we determined the minimum inhibitory concentration (MIC) of boscalid as opposed to the effective concentration that inhibits mycelial growth to 50% of the control. Isolates from nontreated trees had MIC values ranging from 0.1 to 0.5 µg ml-1; the MIC of isolates from commercial orchards ranged from 0.1 to >25 µg ml-1, and isolates from the research orchard ranged from 2.5 to >25 µg ml-1. Isolates with MIC values ≥25 µg ml-1 were considered boscalid resistant and comprised 0% of the nontreated isolates, 30.4% of the commercial isolates, and 42.1% of the research orchard isolates. Sequencing of the sdhB gene of resistant isolates led to the detection of the amino acid mutation H260R, which is known to confer boscalid resistance in other phytopathogenic fungi. Our results indicate that the occurrence of the H260R mutation in Michigan populations of B. jaapii is correlated with the reduction in sensitivity to boscalid observed in commercial orchards.

Evaluation of dodine, fluopyram and penthiopyrad for the management of leaf spot and powdery mildew of tart cherry, and fungicide sensitivity screening of Michigan populations of Blumeriella jaapii.

BACKGROUND: Field trials were conducted on the tart cherry cultivar Montmorency to evaluate the efficacy of dodine and the succinate dehydrogenase inhibitor (SDHI) fungicides fluopyram and penthiopyrad for control of cherry leaf spot (CLS) and powdery mildew (PM). The in vitro sensitivity of Blumeriella jaapii (CLS) to the same fungicides was also tested. RESULTS: Treatments with dodine or fluopyram were among the most effective for controlling CLS, while fluopyram or penthiopyrad treatments were among the most effective for controlling PM. In vitro studies detected a wide range of minimum inhibitory concentrations (MICs) among the isolates (0.05-400 µg AI mL(-1) ) in response to dodine. Orchard isolates showed reduced sensitivity to dodine as compared with baseline isolates. B. jaapii was more sensitive to fluopyram (0.01-10.0 µg AI mL(-1) ) than to penthiopyrad (0.01-25 µg AI mL(-1) ), and orchard isolates also showed a shift towards reduced sensitivity. CONCLUSION: The results indicate that dodine remains effective in CLS control. In addition, as penthiopyrad and fluopyram become available to growers, this research establishes baseline information that will be important for future monitoring and analysis of B. jaapii population responses to exposure to dodine and these SDHI fungicides.

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.

Integration of Copper-Based and Reduced-Risk Fungicides for Control of Blumeriella jaapii on Sour Cherry.

Practical resistance to sterol demethylation inhibitor (DMI) fungicides among populations of Blumeriella jaapii, the cherry leaf spot (CLS) pathogen, was documented in 2005. In the present study, strategies to reduce selection for DMI-resistant strains of B. jaapii and adapt to possible restrictions on the use of chlorothalonil are described. Ten field trials were conducted on the sour cherry cultivars Balaton and Montmorency to test the efficacy of integrating respiration-inhibitor and copper-based fungicides into spray programs. Programs that included up to three sprays of copper-based fungicides were among the most effective for controlling CLS, although leaf phy-totoxicity was sometimes observed. Under high disease pressure, eliminating chlorothalonil compromised CLS control. 'Balaton' and 'Montmorency' did not differ in the percentage of leaves with CLS or defoliation resulting from CLS. The physical modes of action of representative DMI, QoI, and copper-based fungicides were evaluated in a leaf disk assay. Trifloxystrobin, a QoI fungicide, provided the best protection against infection by B. jaapii. All fungicides were more effective than water when applied 46 h postinfection, although differences were not statistically significant in one of two trials. Tebuconazole, a DMI, was the only fungicide that was more effective than water in preventing resporulation from existing lesions in both trials. Isolates of B. jaapii, which varied in DMI-sensitivity, all were sensitive to copper in vitro.

Overexpression of the 14alpha-demethylase target gene (CYP51) mediates fungicide resistance in Blumeriella jaapii.

Sterol demethylation inhibitor (DMI) fungicides are widely used to control fungi pathogenic to humans and plants. Resistance to DMIs is mediated either through alterations in the structure of the target enzyme CYP51 (encoding 14alpha-demethylase), through increased expression of the CYP51 gene, or through increased expression of efflux pumps. We found that CYP51 expression in DMI-resistant (DMI(R)) isolates of the cherry leaf spot pathogen Blumeriella jaapii was increased 5- to 12-fold compared to that in DMI-sensitive (DMI(S)) isolates. Analysis of sequences upstream of CYP51 in 59 DMI(R) isolates revealed that various forms of a truncated non-long terminal direct repeat long interspersed nuclear element retrotransposon were present in all instances. Similar inserts upstream of CYP51 were not present in any of 22 DMI(S) isolates examined.

Occurrence, Distribution, and Polymerase Chain Reaction-Based Detection of Resistance to Sterol Demethylation Inhibitor Fungicides in Populations of Blumeriella jaapii in Michigan.

ABSTRACT The intensive use of site-specific fungicides in agricultural production provides a potent selective mechanism for increasing the frequency of fungicide-resistant isolates in pathogen populations. Practical resistance occurs when the frequency and levels of resistance are great enough to limit the effectiveness of disease control in the field. Cherry leaf spot (CLS), caused by the fungus Blumeriella jaapii, is a major disease of cherry trees in the Great Lakes region. The site-specific sterol demethylation inhibitor fungicides (DMIs) have been used extensively in the region. In 2002, CLS control failed in a Michigan orchard that had used the DMI fenbuconazole exclusively for 8 years. That control failure and our observations from around the state suggested that practical resistance had developed in B. jaapii. Field trial data covering 1989 to 2005 for the DMIs fenbuconazole and tebuconazole supported observations of reduced efficacy of DMIs for controlling CLS. To verify the occurrence of fungicide-resistant B. jaapii, monoconidial isolates were collected in two surveys and tested using a fungicide-amended medium. In one survey, 137 isolates from sites with different DMI histories (no known history, mixed or alternated with other fungicides, and exclusive use) were tested against 12 concentrations of fenbuconazole, tebuconazole, myclobutanil, and fenarimol. Isolates from sites with no prior DMI use were DMI sensitive (DMI(S) = no colony growth at 0.2 mug/ml a.i.) whereas the isolates from the site with prior exclusive use showed growth at DMI concentrations 3 to >100 times higher, and were rated as DMI resistant (DMI(R)). A second survey examined 1,530 monoconidial isolates, including 1,143 from 62 orchard sites in Michigan, where DMIs had been used to control CLS. Resistance to fenbuconazole was detected in 99.7% of the orchard isolates. All isolates from wild cherry trees were sensitive and isolates from feral and dooryard trees showed a range of sensitivities. A polymerase chain reaction (PCR)-based detection method for identifying B. jaapii and DMI(R) was developed and tested. The species-specific primer pair (Bj-F and Bj-R) based on introns in the CYP51 gene of B. jaapii, and the DMI(R)-specific primer pair (DMI-R-Bj-F and DMI-R-Bj-R) based on an insert found upstream of CYP51 in all DMI(R) isolates, provided an accurate and rapid method for detecting DMI(R) B. jaapii. The PCR-based identification method will facilitate timely decision making and continued monitoring of DMI(R) subpopulations in response to management programs.