Thesis
Development of a portable LAMP assay to detect Neofabraea perennans of apple fruit
Washington State University
Master of Science (MS), Washington State University
07/2019
DOI:
https://doi.org/10.7273/000004128
Handle:
https://hdl.handle.net/2376/124655
Abstract
Bull’s eye rot (BER) is a major economic postharvest disease of apple and pear that can be caused by four Neofabraea species i.e., N. perennans, N. alba, N. malicorticis and N. kienholzii. In the growing regions of Central Washington, BER is predominantly caused by N. perennans. The fungus infects fruit preharvest, and because of the dry growing season in the PNW, infections remain latent with symptoms expressed only after 3-4 months of storage, when incidences as high as 20% can been seen in rainy seasons, especially on susceptible cultivars. To detect the infections more efficiently and quickly before symptoms become visible at point-of-care locations, a portable diagnostic tool termed LAMP, Loop mediated isothermal amplification, was developed for early detection of N. perennans. The LAMP technique was optimized and tested for specificity and sensitivity using DNA extracted from pure culture and was used to analyze apple fruits inoculated with N. perennans at different concentrations and sampling times pre- and postharvest. Results indicate that LAMP detected DNA of N. perennans at concentrations as low as 0.001 ng/[mu]l after only 10 minutes of running the reaction. Further validation of the LAMP assay using a portable thermocycler (Genie II®), indicated that LAMP can detect the fungus on apples inoculated with spore suspensions at concentrations of 103 spores/ml or higher prior to commercial maturity without the need for DNA extraction, as well as detecting variability between samples. The assay was further validated using commercial samples of Pinova apples from organic and conventional orchards, demonstrating the ability of this technique to amplify N. perennans from asymptomatic fruit in a commercial setting. LAMP has potential to be used at point of care facilities to better inform the management of BER as fruit goes from harvest into storage, and it has potential to be utilized to better understand the life-cycle of the pathogen.
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Details
- Title
- Development of a portable LAMP assay to detect Neofabraea perennans of apple fruit
- Creators
- David Alexander Enicks
- Contributors
- Achour Amiri (Advisor) - Washington State University, Plant Pathology, Department of
- Awarding Institution
- Washington State University
- Academic Unit
- Plant Pathology, Department of
- Theses and Dissertations
- Master of Science (MS), Washington State University
- Publisher
- Washington State University
- Identifiers
- 99900890782001842
- Language
- English
- Resource Type
- Thesis