Specific and sensitive detection of the conifer pathogen Gremmeniella abietina by nested PCR

Qing-Yin Zeng¹^,2 , Per Hansson³ and Xiao-Ru Wang¹

¹National Institute for Working Life, SE-90713 Umeå, Sweden

²Department of Molecular Biology, Umeå University, SE-90187 Umeå, Sweden

³Department of Silviculture, the Swedish University of Agricultural Sciences, SE-90183 Umeå, Sweden

author email corresponding author email

BMC Microbiology 2005, 5:65doi:10.1186/1471-2180-5-65


Published:	9 November 2005

Abstract

Background

Gremmeniella abietina (Lagerb.) Morelet is an ascomycete fungus that causes stem canker and shoot dieback in many conifer species. The fungus is widespread and causes severe damage to forest plantations in Europe, North America and Asia. To facilitate early diagnosis and improve measures to control the spread of the disease, rapid, specific and sensitive detection methods for G. abietina in conifer hosts are needed.

Results

We designed two pairs of specific primers for G. abietina based on the 18S rDNA sequence variation pattern. These primers were validated against a wide range of fungi and 14 potential conifer hosts. Based on these specific primers, two nested PCR systems were developed. The first system employed universal fungal primers to enrich the fungal DNA targets in the first round, followed by a second round selective amplification of the pathogen. The other system employed G. abietina-specific primers in both PCR steps. Both approaches can detect the presence of G. abietina in composite samples with high sensitivity, as little as 7.5 fg G. abietina DNA in the host genomic background.

Conclusion

The methods described here are rapid and can be applied directly to a wide range of conifer species, without the need for fungal isolation and cultivation. Therefore, it represents a promising alternative to disease inspection in forest nurseries, plantations and quarantine control facilities.