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United States Department of Agriculture

Agricultural Research Service

Related Topics

Research Project: Methods for Rapid Identification and Functional Analysis of Fungi Causing Postharvest Decay of Pome Fruit

Location: Food Quality Laboratory

2013 Annual Report

1a. Objectives (from AD-416):
Objective 1: Isolate and identify fungi causing postharvest decay of pome fruit from packinghouses that use different pest management programs and determine their levels of resistance to postharvest fungicides. [NP 303; C1, PS 1A] Sub-objective 1A: Determine the tolerance of Penicillium spp., causing blue mold of apples from conventional and organic packinghouses, to postharvest fungicides. Sub-objective 1B: Determine cultural, morphological, and molecular markers that distinguish species and strains of Penicillium that differ in virulence and their sensitivity to selected fungicides. Sub-objective 1C: Design a real-time PCR method for rapid and accurate detection of Penicillium spp. causing blue mold of apple. Objective 2: Design and construct a genetic transformation system for functional analysis of genes in fungi that cause postharvest decay of pome fruit. [NP 303; C2, PS 2A] Sub-objective 2A: Construct binary vectors containing selectable antibiotic resistance gene(s), and transform into Penicillium expansum using Agrobacterium tumefaciens. Sub-objective 2B: Determine the mitotic stability and cultural morphology of Penicillium expansum transformants carrying single and multiple antibiotic resistance genes in vitro. Sub-objective 2C: Assess virulence and morphology in apple fruit of Penicillium expansum transformant strains harboring selectable antibiotic resistance genes. Objective 3: Identify and characterize genes encoding components of pathways that effect pathogen growth or virulence in fungi causing postharvest decay of pome fruit. [NP 303; C2, PS 2A] Sub-objective 3A: Identify signaling pathways and characterize genes that potentially affect conidial germination in Penicillium expansum causing blue mold. Sub-objective 3B: Analyze the function of candidate genes involved in pathways that affect Penicillium expansum growth or virulence.

1b. Approach (from AD-416):
This research aims to devise innovative detection methods for Penicillium spp. and to identify new targets for control to reduce blue mold decay during storage. The project entails the characterization of Penicillium species obtained from decayed apple fruit from packinghouses that use different disease control strategies. Tolerance to postharvest fungicides will be determined, Penicillium isolates will be identified to species, and conserved gene sequences will be used to develop a rapid and accurate detection method. This information will assist in management decisions that will guide the selection and use of postharvest fungicides. A transformation system will be developed to evaluate virulence gene function in Penicillium expansum. Transformants will be evaluated for aberrations in conidial germination and fungal virulence and genetic analyses will be implemented to uncover novel virulence-related genes. This research is anticipated to provide a pioneering method to analyze gene function while concomitantly uncovering new genes involved in fungal virulence that will serve as specific targets for decay control. Inhibitors and activators of highly conserved signaling pathways will be used to unveil additional genes involved in conidial morphogenesis and fungal virulence in Penicillium spp. A targeted functional genetic analysis of gene candidates will provide new information regarding the mechanisms controlling fungal virulence and conidial germination which may be used to develop targeted decay control strategies.

3. Progress Report:
Penicillium expansum and P. solitum are the most destructive fungi causing blue mold of apples and produce toxins that contaminate processed fruit products like apple cider, juice and sauce. Rapid detection is vital for control and work conducted in the Food Quality Laboratory in Beltsville, Maryland has made significant progress in developing a detection system for P. expansum and P. solitum. Multiple primer pairs were designed for use in both conventional and real time Polymerase Chain Reaction (PCR). One primer set detected multiple Penicillium species, while the other discriminated between P. expansum and P. solitum. The method will be useful to plant diagnosticians for rapid and accurate diagnosis and for packinghouse operators to target blue mold infested areas for sanitation to reduce decay. Chemical controls for P. expansum and P. solitum are limited, and their efficacy is rapidly declining due to fungicide-resistant Penicillium strains. There is an urgent need to develop additional control strategies for Penicillium species to help stakeholders maintain apple fruit quality during storage. Scientists at the Beltsville lab have taken the first step in designing a fungal transformation system to analyze virulence genes in P. expansum and P. solitum. Specific antibiotics were determined to be effective selection agents and corresponding resistance genes were obtained and cloned into commercial plasmids. Specific genes in P. expansum and P. solitum can now be mutated and their role in postharvest decay can be determined using the transformation system. The information can be used to tailor specific control strategies focused on genes that control virulence in blue mold fungi. This research will benefit stakeholders via development of new effective control strategies to reduce blue mold decay during long-term storage. This research directly addresses the project plan’s objectives of developing rapid detection methods and determining the genetic basis of virulence mechanisms for effective and sustainable control measures against blue mold on apple fruit.

4. Accomplishments
1. Discovery of new apple rot fungi in the eastern United States. Three different fungal plant pathogens were found causing postharvest decay on apple fruit from commercial operations located in Pennsylvania and Maryland. These fungi represent emerging pathogens for the Mid Atlantic apple fruit growing area as this is the first time they have been reported. This information will help other scientists develop specific detection methods to rapidly identify these pathogens and also will help apple fruit growers, packers and processors use the most effective practices to control decay during storage.

2. Role of calcium in regulating fruit ripening and resistance to decay. Calcium is a ubiquitous naturally occurring element that is involved in a variety of biological processes in plants, humans and animals. Results from a study using tomato fruits show that calcium-regulated genes are also controlled by plant hormones and by the grey mold fungus during infection. The research knowledge can be used to improve disease resistance against grey mold and help improve fruit quality by altering hormones responsible for fruit ripening.

3. Use of beneficial bacteria to control brown rot on stone fruit. Brown rot is the most destructive disease of plums that causes losses in the field and in storage. Several different naturally occurring bacterial species were obtained from the surface of plum fruit that could be used in place of commercial fungicides to substantially reduce brown rot. Applied as biological control agents for brown rot management on stone fruit, these bacteria have immediate application in organic agriculture postharvest management.

Review Publications
Jurick II, W.M., Vico, I., Gaskins, V.L., Janisiewicz, W.J., Peter, K.A. 2013. First Report of Neofusicoccum ribis causing postharvest decay of apple fruit from cold storage in Pennsylvania. Plant Disease. 97(7):999.

Jurick II, W.M., Vico, I., Gaskins, V.L., Janisiewicz, W.J., Peter, K.A. 2013. First report of Botryosphearia dothidea causing white rot on apple fruit in Maryland. Plant Disease. DOI: 10.1094/PDIS-01-13-0053-PDN.

Peter, K.A., Vico, I., Gaskins, V.L., Janisiewicz, W.J., Saftner, R.A., Jurick II, W.M. 2012. First report of Penicillium carneum causing blue mold on stored apples in the United States. Plant Disease. 96:1823.

Janisiewicz, W.J., Jurick II, W.M., Vico, I., Peter, K.A., Buyer, J.S. 2012. Culturable bacteria from plum fruit surfaces and their potential for controlling brown rot after harvest. Postharvest Biology and Technology. 76:145-151.

Last Modified: 2/23/2016
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