PCR detection of Gremmeniella abietina, the causal agent of Scleroderris canker of pine

Abstract

Scleroderris canker of conifer is caused by Gremmeniella abietina var. abietina, which comprises several taxa, including races, varieties, and biotypes. The European race of G. abietina var. abietina was introduced into North America early in the century, most likely on asymptomatic infected pine seedlings, and is currently widespread in eastern North America. In order to detect latent infections and differentiate the North American and European races of the fungus, we developed oligonucleotide primers to amplify by PCR portions of the ITS of the ribosomal DNA of G. abietina var. abietina. The 417 bp amplified DNA fragment comprises two Msp I restriction sites in the NA race but only one in the EU race. DNA extractions directly from infected asymptomatic needles, or from single fruiting bodies, followed by PCR amplification and Msp I digestion allowed the detection and race identification of both races of G. abietina var. abietina from seedlings and branches of Pinus resinosa and P. banksiana. A nested PCR assay was sensitive enough to detect the equivalent of a single infected seedling in a bulk sample of 1000 healthy seedlings. Validation tests were conducted by comparing PCR and isolation assays with 104 fascicles. All samples for which the fungus was isolated yielded a positive PCR assay and there was no false negative, i.e. samples for which the fungus was isolated but not detected by PCR. Among the samples from which the fungus was not isolated, most yielded a negative PCR assay (71%), but a proportion (29%) yielded positive PCR assays. In several of those cases, aggressive contaminants had apparently overgrown the pathogen. The method described here can lead to the detection and race identification of the NA and EU races of G. abietina var. abietina directly from infected tissues without the need to culture the fungus and should find applications in nursery inspection and quarantine.