Location: Food Quality Laboratory
2021 Annual Report
Objectives
Objective 1: Identify key genes regulating virulence and toxin production in Penicillium species, the causal agents of blue mold, to develop novel gene or protein targets for control in commercially stored pome fruit.
Sub-objective 1.A: Identify new Penicillium spp. virulence and toxin biosynthetic genes via comparative genomics and transcriptomics.
Sub-objective 1.B: Characterize fungal virulence and toxin genes in Penicillium spp. using a targeted gene deletion approach.
Objective 2: Integrate genomic-based strategies and evaluate novel tools to manage postharvest blue mold decay in commercial storage caused by Penicillium species on pome fruit.
Sub-objective 2.A: Determine difenoconazole baseline sensitivity and characterize resistant blue mold isolates.
Sub-objective 2.B: Identify Penicillium spp. genes associated with difenoconazole resistance and develop a molecular-based detection system.
Approach
Multiple approaches are outlined in this project that encompass both basic and applied methodologies to maintain pome fruit quality, deliver effective strategies to manage blue mold decay, and eliminate mycotoxins from processed pome fruit products. Comparative genomics and transcriptome sequencing will be used to discover new fungal virulence genes and pathways that regulate Penicillium spp. virulence, and toxin production to develop pathogen-specific management strategies. Additionally, mechanisms of postharvest fungicide resistance in Penicillium spp. will be determined using a genomics approach to develop molecular-based management tools for producers. Our applied research focus will utilize standard microbiological methods to determine baseline sensitivity to a new postharvest fungicide currently used to manage blue mold decay and will help producers monitor future shifts in sensitivity indicative of resistance. Characterization of fungicide-resistant isolates will provide practical information on the viability and persistence of such isolates in the packing and storage environments and their impact on control using currently available chemical tools labeled for pome fruits. Results from the current study will also guide growers in making decisions for use of the most efficacious fungicides to control blue mold.
Progress Report
The current project has generated innovative and practical accomplishments which have impacted our stakeholders and customers in the scientific community and the pome fruit industry. Publications have appeared in high-impact, peer-reviewed journals, invited review articles, trade journals, and book chapters. Their titles alone offer insight into the basic, applied, and translational research that was conducted over a 5-year cycle. Under Objective 1, the genetic identity of multiple, independent T-DNA mutants with virulence defects in the apple was accomplished. Further characterization of one of these mutants led to the discovery of the first master regulatory gene in fungi (Blistering1) that controls decay, toxin production, and CAZyme secretion. New omics-based resources, including genomic, transcriptomic, and proteomic resources for wild type, Blistering1 mutants, and fungicide resistant isolates, were generated and released to the public. Additional dominant selectable markers for blue mold fungal transformation were also discovered and analyzed. For Objective 2, baseline sensitivity and a discriminatory dose for monitoring resistance to difenoconazole, the active ingredient in postharvest fungicide Academy, in Penicillium spp. was generated. Whole annotated blue mold fungal genomes and transcriptomes for difenoconazole sensitive strain (P11) and difenoconazole resistant (G10) were assembled, annotated, and released. A global mechanism for difenoconazole resistance in Penicillium spp. was elucidated, and new tools for detecting resistant isolates were developed and tested. Other significant achievements include developing and validating a rapid bioassay to assess patulin mode of action in planta and on phytopathogenic fungi. Quality genomic DNA was isolated from multiple Colletotrichum spp. that cause bitter rot on apples and was used for whole-genome sequencing, assembly, and annotation. Thus, producing the first set of bitter rot genomes from the Mid Atlantic area. Genomic resources for the apple scab fungus, Venturia inequalis, with fungicide resistance phenotypes, were produced and deposited in a publicly accessible database. These new fungal genomic resources have helped refine species-level phylogeny, aided in the development of nucleic acid detection strategies, and helped to ascertain mechanisms of virulence and fungicide resistance that were previously not possible.
Accomplishments
1. Antimicrobial Resistance Risk Prediction/Management Tool. Fungicides are the primary tools to control postharvest fungal pathogens, and antimicrobial resistance is a widespread problem that has reduced efficacy. ARS researchers in Beltsville, Maryland, in collaboration with the University of Washington, were the first to show that there is a significant correlation between a mutation in the ß-tubulin gene and the year an isolate was collected, the pathogen genus, the host, and the collection region for FRAC1 fungicides. The publicly available dataset can help inform stakeholders and customers on specific regions and hosts most likely to contain fungicide-resistant phytopathogens. Thus, assisting in decisions concerning fungicide resistance management can be used to make inferences concerning the probability and speed at which resistance develops in a fungal population.
Review Publications
Litchner, F.J., Gaskins, V.L., Cox, K., Jurick II, W.M. 2020. Global transcriptomic responses orchestrate difenoconazole resistance in Penicillium spp. causing blue mold of stored apple fruit. BioMed Central (BMC) Genetics. https://doi.org/10.1186/s12864-020-06987-z.
Luciano-Rosario, D., Keller, N.P., Jurick II, W.M. 2020. Penicillium expansum: biology, omics, and management tools for a global postharvest pathogen causing blue mold of pome fruit. Molecular Plant Pathology. 21:1391–1404. https://doi.org/10.1111/mpp.12990.
Bartholomew, H.P., Bradshaw, M., Fonseca, J.M., Jurick II, W.M. 2021. The good, the bad and the ugly: mycotoxin production during postharvest decay and their influence on tritrophic host-pathogen-microbe interactions. Frontiers in Microbiology. 12:1-12. https://doi.org/10.3389/fmicb.2021.611881.
Bradshaw, M., Bartholomew, H., Fonseca, J.M., Gaskins, V.L., Prusky, D., Jurick II, W.M. 2021. Delivering the goods: Fungal secretion modulates virulence during host-pathogen interactions. Fungal Biology Reviews. 36:76-86. https://doi.org/10.1016/j.fbr.2021.03.007.
