SECTION 1: ABSTRACT Aster yellows phytoplasma (AYP) is a phloem-limited bacterial plant pathogen affecting garlic production in Minnesota. Because phytoplasmas are difficult to culture, diagnosis relies mainly on laboratory-based PCR, qPCR, and sequencing, which limits rapid field decision-making for growers. The overall goal of this project is to develop a proof-of-concept, field-deployable CRISPR-Cas12a diagnostic workflow for detecting phytoplasma and AYP in garlic. I hypothesize that combining simplified sample preparation, RPA isothermal amplification, CRISPR-Cas12a detection, and lateral-flow or fluorescence readout can enable faster plant disease detection while maintaining sensitivity comparable to qPCR. The first aim is to design and computationally evaluate RPA primers and Cas12a crRNAs targeting cpn60 and ribosomal protein (RP) sequences generated from phytoplasma-positive garlic samples. The second aim is to validate the CRISPR-Cas12a detection workflow using amplified targets and extracted nucleic acids, including preliminary testing with three literature-derived crRNAs that produced detectable fluorescence signal in phytoplasma-positive targets. The third aim is to translate the assay toward a portable detection platform using multiplex lateral flow and future probe-invasion logic. Methods include multilocus sequence analysis, crRNA design in Google Colab, BLAST/NUPACK screening, RPA, Cas12a reporter assays, LOD estimation, qPCR comparison, and field-compatible sample preparation optimization.
