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Molecular detection and quantification of leaf rust spores in wild blueberry.

Nghi Nguyen: University of Maine

<div>Microscopy has traditionally been incorporated with spore trap sampling to detect and quantify spores of fungal pathogens that cause wild blueberry leaf spots in Maine. However, this method is laborious and requires a great deal of technical training. To improve spore detection and quantification, quantitative polymerase chain reaction (qPCR) and quantitative loop mediated isothermal amplification (qLAMP) are being developed for <em>Thekopsora minima, </em>which causes leaf rust in wild blueberry. Rust urediniospores were collected from four different fields in Maine, from which DNA was extracted and then PCR was used to amplify the internal transcribed spacer (ITS) regions of ribosomal DNA. The amplicons were sequenced and analyzed to develop primers specific to <em>T. minima</em>. Six potential primer sequences unique to rust in the ITS regions were selected. One primer pair showed specificity for rust compared to other fungal species and was optimized for its detection limit and for qPCR. These specific primers will be modified or developed anew for qLAMP. The molecular methods will be compared to microscopy for spore quantification using spore traps and then tested for their efficacy in detecting early infection in plants. Early detection in plants and better spore counts will be coupled with weather data to determine factors affecting spore dispersal and infection to develop a more efficient forecasting system for leaf rust in blueberry.</div>