Use of Sodium Hydroxide DNA Extraction Methods for Nested PCR Detection of Bretziella fagacearum in the Sapwood of Oak Species in Minnesota

Abstract

Oak wilt caused by Bretziella fagacearum is an important disease of Quercus spp.; however, its diagnosis may be confused with damage resulting from other diseases, insects, or abiotic factors. Laboratory diagnosis is important in such situations and when disease control action is desired. PCR tests can provide accurate lab diagnosis within 2 days. Two variations of a simple DNA extraction protocol using sodium hydroxide (NaOH) were compared with that of the proprietary protocol of a commercially available kit (CK) for nested PCR to detect the pathogen in oak sapwood. High frequencies of pathogen detection (98 to 100% of 48 branch segments assayed) were found for northern pin oak using the two NaOH-based and the CK methods. Detection rates were similar but lower for bur oak (ranged from 58 to 79%) and white oak (ranged from 54 to 71%) regardless of DNA extraction method. Using our alternative DNA extraction protocols may reduce total time and cost of B. fagacearum detection in PCR-based diagnosis and other downstream applications.