Development of a Portable LAMP Assay for Detection of Neofabraea perennans in Commercial Apple Fruit.

Bull's eye rot (BER) is a major economic postharvest disease of apple and pear that can be caused by four Neofabraea species: N. perennans, N. alba, N. malicorticis, and N. kienholzii. In Central Washington, BER is predominantly caused by N. perennans. The fungus infects fruit preharvest, and because of the dry growing season in the region, infections remain latent with symptoms expressed only after 3 to 4 months of storage, when BER incidences as high as 20% can been seen, especially in rainy seasons and on susceptible cultivars. To ensure early and efficient infection detections before BER symptoms become visible at point-of-care locations, a portable diagnostic tool based on loop-mediated isothermal amplification (LAMP) was developed using the ß-tubulin gene. The LAMP assay was optimized and tested for specificity and sensitivity using DNA extracted from pure cultures of N. perennans and seven other fungal species. The results showed that the selected LAMP primer set was specific to N. perennans and highly sensitive as it detected DNA concentrations as low as 0.001 ng/µl after only 10 min. The assay was validated for N. perennans detection on artificially inoculated apples using a portable thermocycler, Genie II, without the need for DNA extraction. The LAMP assay detected N. perennans on apples inoculated with spore suspensions 3 weeks prior to harvest at concentrations of 103 spores/ml or higher. The assay was further validated using commercial Piñata apples from organic and conventional orchards, demonstrating the ability of this technique to amplify N. perennans from asymptomatic fruit in a commercial setting 3 months before commercial maturity. The LAMP assay developed for N. perennans detection can be easily expanded to detect the other BER causal species. LAMP has potential to be used in orchards and at point-of-care facilities to better inform on BER management at different fruit growth stages, and it has potential to be utilized to better understand the epidemiology of Neofabraea spp.

On-Site Molecular Detection of Soil-Borne Phytopathogens Using a Portable Real-Time PCR System.

On-site diagnosis of plant diseases can be a useful tool for growers for timely decisions enabling the earlier implementation of disease management strategies that reduce the impact of the disease. Presently in many diagnostic laboratories, the polymerase chain reaction (PCR), particularly real-time PCR, is considered the most sensitive and accurate method for plant pathogen detection. However, laboratory-based PCRs typically require expensive laboratory equipment and skilled personnel. In this study, soil-borne pathogens of potato are used to demonstrate the potential for on-site molecular detection. This was achieved using a rapid and simple protocol comprising of magnetic bead-based nucleic acid extraction, portable real-time PCR (fluorogenic probe-based assay). The portable real-time PCR approach compared favorably with a laboratory-based system, detecting as few as 100 copies of DNA from Spongospora subterranea. The portable real-time PCR method developed here can serve as an alternative to laboratory-based approaches and a useful on-site tool for pathogen diagnosis.