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.

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.

First report of foliicolous fungus Trichothecium roseum (Pers.) Link on sweet cherry Prunus avium L. from Kashmir Valley

Cherry Prunus avium L. is an important cash crop of Kashmir Valley. Cherry leaves are attacked by number of foliicolous fungi which in turn reduce yield of crop. Severe leaf spots were observed on cherry leaves in an orchard of district Baramulla of Kashmir Valley. These spots were similar to those caused by Blumeriella jaapii but smaller in size. Pathogenicity tests were conducted using detached leaf technique. Re isolation of spores from inoculated leaves confirmed Trichothecium roseum as causative agent of purple leaf spot of cherry. This is the first report of Trichothecium roseum responsible for leaf spot of cherry in Kashmir Valley.

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.

Screening Ornamental Cherry (Prunus) Taxa for Resistance to Infection by Blumeriella jaapii

Ornamental flowering cherry trees are important landscape plants in the United States but are susceptible to several serious pests and disease problems. Cherry leaf spot, incited by the fungus Blumeriella jaapii, is characterized by defoliating susceptible trees in late summer, leading to weakening or even death of the tree. To identify resistant plants for use in landscape plantings and in our breeding program, we used a detached leaf assay to screen 69 diverse ornamental flowering cherry taxa for resistance to cherry leaf spot. We found clear differences in susceptibility among the accessions, with seven accessions developing essentially no symptoms at all. A variance decomposition showed that most of the variance (59%) occurred among accessions, indicating that genotype, even more than species, determined susceptibility. The detached leaf assay used in this study is an effective method for screening large numbers of plants for relative resistance to cherry leaf spot. These methods will be particularly useful to characterize germplasm and screen hybrids in breeding and selection programs.

Investigation of biological peculiarities of Blumeriella jaapii

Leaf spot pathogen is characterized as a highly developed parasite after investigation of development peculiarities of the fungus in pure culture and natural conditions. The degree of correlation depended on the medium. Impact of incubation temperature on Blumeriella jaapii (Rehm) Arx growth was different. Fungi colonies formed more intensively and their diameter increased significantly under higher temperature. Optimal conditions for the disease prevalence are 15–20°C and moisture. Relative humidity and precipitation influenced maturation of ascomycetes and discharge of ascospores (r = +0.89). Microscopic analysis of leaves showed that during winter thaw, when the average temperature is about 0 ± 5°C and relative humidity is higher than 70%, was possible to detect mature ascomycetes and conidia. It is possible to affirm that lower temperature than it was assumed is sufficient for formation of ascomycetes.