Real-Time PCR Assays to Detect and Distinguish the Rapid 'Ōhi'a Death Pathogens Ceratocystis lukuohia and C. huliohia.

Ceratocystis lukuohia and C. huliohia are recently described fungal species that cause rapid 'ōhi'a death (ROD) of Metrosideros polymorpha, Hawaii's most abundant and ecologically important native species. Although the pathogens are now widespread on Hawai'i Island, a major effort is underway to study and manage affected forests, and particularly to prevent the disease from spreading to other islands in the State or throughout the Pacific. Rapid and accurate detection is critical. Molecular diagnostic real-time PCR protocols were developed to detect and distinguish the two pathogens, suitable for detection of fungal DNA from extracts of wood, soil, and insect frass. The assays detect as few as 2 to 4 or 16 spores of C. huliohia or C. lukuohia, respectively. These assays are valuable tools for monitoring disease spread and offer a significant advantage over culture-based methods for diagnostics, requiring <1 day to arrive at definitive results.