Location: Food Quality Laboratory2019 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.
The following progress has been accomplished during FY19 for both research objectives of the project plan. Under Objective 1, which aims to identify and verify genes encoding fungal virulence factors in the blue mold fungus, we have uncovered a novel global regulator mediating decay and toxin production. The single copy gene encodes a small polypeptide with a DNAJ domain, but no other conserved motifs. Studies are ongoing to identify associated interacting partners to provide additional candidates for targeted blue mold control. Comparative transcriptome sequencing of 2 Penicillium spp. isolates with different levels of virulence in apple fruit has been executed and differential gene expression analyses are being conducted. Whole genome comparisons between multiple Penicillium spp. has revealed an array of toxin, vitamin, small molecule, and secondary metabolic biosynthetic gene clusters, some of which have not previously been described in Penicillium spp. and may be of biotechnological and/or pharmacological interest. Under Objective 2, which seeks to determine the mechanism(s) of fungicide resistance to design tools for detecting resistant strains, a comparative transcriptomics approach has been executed between an azole-resistant and azole-sensitive blue mold isolates. Differential gene expression (DGE) analysis revealed overexpression of Cytochrome P450 (CYP51) in the resistant isolate compared to the sensitive strain. The resistant isolate has multiple single amino acid changes in CYP51 conserved domains which may confer resistance to difenoconazole. Our detailed DGE studies have also uncovered a variety of multi-drug efflux pumps and transporters that may facilitate excretion of the fungicide. Hence, we are verifying the role of these pumps and conducting single nucleotide polymorphism calling of differentially expressed genes to generate rapid molecular markers to monitor and abate antimicrobial resistance to azole fungicides in Penicillium spp.
1. Developing tools to combat antimicrobial resistance in the blue mold fungus. Apples are one of the most consumed fruit in the U.S. which are stored for 6 to 12 months. During such time, the fruit become increasingly susceptible to rot caused by the blue mold fungus that reduces quality, contributes to waste, and produces the mycotoxin patulin. A new postharvest fungicide for apple fruit was released for use in 2015. ARS researchers in Beltsville, Maryland, in collaboration with Cornell University determined the sensitivity to patulin of a globally diverse Penicillium spp. population that cause blue mold, developed a discriminatory dose to monitor antimicrobial resistance, and tested the patulin formulation against different fungicide resistant blue mold isolates. A discriminatory dose for difenoconazole is being used by extension professionals to detect fungicide resistant strains to maintain efficacy of current postharvest chemicals in the Mid-Atlantic and Pacific Northwest regions.
Jurick II, W.M., Macarisin, O., Gaskins, V.L., Janisiewicz, W.J., Peter, K.A., Cox, K.D. 2018. Baseline sensitivity of Penicillium spp. to difenoconazole. Plant Disease. 103:331-337. http://doi.org/10.1094/PDIS-05-18-0860-RE.
Wu, G., Jurick II, W.M., Yin, G., Yu, J., Peng, H., Gaskins, V.L., Yin, Y., Hua, S.T., Peter, K., Shelton, D.R. 2019. Whole-genome comparisons of Penicillium spp. reveals secondary metabolic gene clusters and candidate genes associated with fungal aggressiveness during apple fruit decay. PeerJ. 7:e6170. https://doi.org/10.7717/peerj.6170.