Specific detection of Waiteacircinata var.zeae using conventional and real-time PCR

Abstract

Waiteacircinata var. zeae, a pathogen with a relatively narrow host range, has recently been detected in cabbage and oilseed rape in Europe and worldwide. In this study, we developed specific conventional and real-time PCR protocols for direct detection of W.circinata var. zeae from mycelium and diseased plant tissue. The newly developed primer pair zeaefor1/zeaerew1, used in PCR protocols, specifically amplified only target isolates of W.circinata var. zeae when tested against isolates of 11 different binucleate and multinucleate anastomosis groups of Rhizoctonia spp. including AG-A, AG-G, AG-F, AG-U, AG-2-1, AG-2-2, AG-3, AG-4 HGI, AG-4 HGII, AG-4 HGIII, and AG-6 and common soil-borne pathogens. Total of nine previously published primer pairs designed for the detection of various Rhizoctonia spp. were also tested and did not amplify target isolates of W.circinata var. zeae. The detection limit of conventional and real-time PCR protocols was 10(-2) and 10(-5) (with starting concentration 9.5 ng/mu l), respectively, and both methods are the first available tools for direct detection and identification of W.circinata var. zeae from mycelium and diseased oilseed rape seedlings. Both conventional and SYBR-Green-based real-time PCR protocols are cost-effective and provide a solid basis for further investigations of W.circinata var. zeae, particularly in relation to distribution, host range, and epidemiology.