Location: Food Quality Laboratory2020 Annual Report
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.
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 has been made during FY20 for both research objectives of the USDA-ARS in house research project plan. Under Objective 1, which aims to identify and verify genes encoding fungal virulence factors in blue mold fungi, we have generated 4 independent single gene deletion mutants in the Penicillium expansum blistering1 locus. We have characterized these mutants with respect to their morphology, growth, conidial germination rate, and virulence in apple fruit. Their phenotype of these mutants closely mirrors that of a random P. expansum T-DNA insertional mutation in the coding region of the blistering1 gene. These findings allow for research to proceed forward on this master regulator to dissect interacting proteins and pathways to develop blue mold-specific controls. Comparative RNAseq analysis of ungerminated conidia and decayed apple fruit of 2 Penicillium spp. with different levels of aggressiveness are underway and expected to reveal a plethora of genes involved in decay. These genes will be subjected to mutational analysis to verify their role in decay and will reveal loci crucial for spore survival and germination. Objective 2 seeks to determine the mechanism(s) of antimicrobial resistance and to design tools for detecting resistant strains. It was discovered that the blue mold fungus possesses three different Cytochrome P450 monoxygenase genes (CYP51A, B, and C). Real time PCR expression of the three genes was carried out in the resistant and sensitive strains and differential expression was observed after difenoconazole exposure. Examination of promoter regions of each gene did not reveal any transposons, repetitive elements, or signatures associated with azole fungicide resistance. Studies were conducted showing that multi-drug efflux pumps control resistance to difenoconazole in vitro using broad spectrum pump inhibitors. Knowledge of these pumps affords a new avenue to develop materials that can chemo sensitize resistant isolates to facilitate blue mold control with existing fungicides.
1. Stopping blue mold fungus decay in apples. Apples are one of the most popular consumed fruits in the U.S., which may be in storage for up to 12 months. During storage, the blue mold fungus may cause the apple to rot, reducing its quality. Blue mold is difficult to control, and new methods are needed. ARS researchers in Beltsville, Maryland, in collaboration with the University of Wisconsin, Penn State University, and Dartmouth University, have discovered a way to block the gene in the blue mold fungus that causes apple rot in storage. The apple industry and researchers are using this new knowledge to develop postharvest decay treatments for the blue mold fungus in apples.
Jurick II, W.M., Peng, H., Beard, H.S., Garrett, W.M., Macarisin, O., Peter, K., Gaskins, V.L., Yang, T., Lu, Y., Mowery, J.D., Bauchan, G.R., Cooper, B. 2019. Blistering1 modulates Penicillium expansum virulence via vesicle-mediated protein secretion. Molecular and Cellular Proteomics. https://doi.org/10.1074/mcp.RA119.001831.
Lichtner, F.J., Jurick II, W.M., Ayer, K.M., Gaskins, V.L., Villani, S.M., Cox, K.D. 2019. Venturia inaequalis genomes with multiple fungicide resistance phenotypes causing preharvest apple scab and postharvest pinpoint scab. Phytopathology. https://doi.org/10.1094/PHYTO-06-19-0222-A.
Liu, Y., Oh, S., Jurick II, W.M. 2019. Response of Aspergillus flavus spores to nitric oxide fumigations in atmospheres with different oxygen concentrations. Journal of Stored Products Research. 83:78-83. https://doi.org/10.1016/j.jspr.2019.06.001.