Exploring CRISPR-Based Detection of Lecanosticta acicola in Loblolly Pine: From DNA Extraction to Field-Forward Diagnostics for Brown Spot Needle Blight

Abstract

Brown spot needle blight (BSNB), caused by Lecanosticta acicola, reduces growth and may contribute to mortality in loblolly pine (Pinus taeda), but diagnosis is complicated by symptom overlap with other needle diseases and by inhibitor-rich conifer tissues. This thesis presents an exploratory evaluation of CRISPR-based diagnostics for L. acicola, with a particular focus on the pre-analytical steps that determine whether sufficient pathogen DNA can be reliably recovered from BSNB-infected needles. Chapter 1 reviews classical, molecular, and point-of-care diagnostic approaches for woody plant pathogens and highlights CRISPR-Cas platforms as a promising bridge between laboratory-level specificity and field deployment. Chapter 2 compares five DNA extraction workflows and six ITS2 amplification conditions, including PNA-based host DNA suppression, and shows that fungal-enriching chemistries combined with selective amplification yield stronger and more consistent detection of L. acicola than plant- or microbiome-focused kits. Chapter 3 builds on these optimized conditions to develop and validate proof-of-concept CRISPR-Cas assays targeting the ITS and TEF1 loci of L. acicola, using in vitro Cas9 ribonucleoprotein cleavage to screen guide RNAs and confirm target suitability for diagnostic application. Together, these results demonstrate that combining extraction strategies that favor fungal DNA with CRISPR-based detection improves diagnostic performance from pine needles and provides a practical foundation for future field-deployable BSNB diagnostic tools.