Improved isolation and PCR detection of Phytophthora agathidicida oospores from soils.

Phytophthora species are eukaryotic microorganisms responsible for severe dieback and root rot in plants worldwide, impacting crops, forests, and other important ecosystems. In New Zealand, P. agathidicida leads to fatal dieback in kauri (Agathis australis), long-lived endemic trees of significant cultural and ecological importance. A critical aspect of the P. agathidicida lifecycle is the production of oospores-thick-walled spores essential for long-term survival in soil, dispersal, and disease inoculation. However, their heterogeneous distribution in soils, robust structure, and dormant state make them challenging to detect using soil baiting or DNA-based methods. Soil baiting is the basis of most current testing for P. agathidicida, but baiting-based methods have low sensitivity, are slow, and require specialised facilities. To address these challenges, we developed and validated a PCR-based method for detecting P. agathidicida oospores directly from soil. Our approach includes a technique for separating oospores from soil, improved oospore lysis and DNA extraction, and a primer pair that targets a repeat region of the P. agathidicida genome with high sensitivity and specificity. The primers amplified the target product in all tested P. agathidicida isolates without cross-reactivity against eight non-target Phytophthora species. The detection limit was 1 femtogram of P. agathidicida DNA via endpoint PCR. Performance assessment against 65 soil samples from kauri forests revealed P. agathidicida in 69% of samples compared to only 11% detected by existing methods. By eliminating the need for baiting, our assay enhances the speed, accuracy, and accessibility of testing, thereby facilitating more comprehensive monitoring and improved disease management.

Importance: Phytophthora species are notorious plant pathogens responsible for severe dieback and root rot diseases, significantly impacting crops, forests, and irreplaceable natural ecosystems. Rapid and accurate detection of these pathogens is essential for effective disease management. In New Zealand, P. agathidicida threatens the country's endemic kauri forests. In this study, we developed and validated a PCR-based method for detecting P. agathidicida oospores in soil. Oospores are long-lived, thick-walled spores that serve as key propagules for survival in soil and the spread of disease. Their robust structure and dormant state make them particularly challenging to detect using traditional soil baiting techniques or DNA-based methods. Our method is fast, accurate, and requires minimal equipment, enabling local testing and thereby empowering communities and enhancing surveillance efforts. Although developed for P. agathidicida, this method could be adapted for other plant pathogens, potentially improving disease management across various agricultural and ecological contexts.