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Applied and Environmental Microbiology, August 2003, p. 4413-4420, Vol. 69, No. 8
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.8.4413-4420.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Multiplex Real-Time PCR for Monitoring Heterobasidion annosum Colonization in Norway Spruce Clones That Differ in Disease Resistance

Ari M. Hietala,^* Morten Eikenes, Harald Kvaalen, Halvor Solheim, and Carl G. Fossdal

Skogforsk, 1432 Ås, Norway

Received 26 December 2002/ Accepted 7 May 2003

A multiplex real-time PCR assay was developed to monitor the dynamics of the Picea abies-Heterobasidion annosum pathosystem. Tissue cultures and 32-year-old trees with low or high resistance to this pathogen were used as the host material. Probes and primers were based on a laccase gene for the pathogen and a polyubiquitin gene for the host. The real-time PCR procedure was compared to an ergosterol-based quantification method in a tissue culture experiment, and there was a strong correlation (product moment correlation coefficient, 0.908) between the data sets. The multiplex real-time PCR procedure had higher resolution and sensitivity during the early stages of colonization and also could be used to monitor the host. In the tissue culture experiment, host DNA was degraded more rapidly in the clone with low resistance than in the clone with high resistance. In the field experiment, the lesions elicited were not strictly proportional to the area colonized by the pathogen. Fungal colonization was more restricted and localized in the lesion in the clone with high resistance, whereas in the clone with low resistance, the fungus could be detected until the visible end of the lesion. Thus, the real-time PCR assay gives better resolution than does the traditionally used lesion length measurement when screening host clones for resistance.

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* Corresponding author. Mailing address: Skogforsk, Høgskoleveien 12, 1432 Ås, Norway. Phone: 47 64949049. Fax: 47 64942980. E-mail: ari.hietala{at}skogforsk.no.

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Applied and Environmental Microbiology, August 2003, p. 4413-4420, Vol. 69, No. 8
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.8.4413-4420.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

This article has been cited by other articles:

• Hietala, A. M., Nagy, N. E., Steffenrem, A., Kvaalen, H., Fossdal, C. G., Solheim, H. (2009). Spatial Patterns in Hyphal Growth and Substrate Exploitation within Norway Spruce Stems Colonized by the Pathogenic White-Rot Fungus Heterobasidion parviporum. Appl. Environ. Microbiol. 75: 4069-4078 [Abstract] [Full Text]  
• Hietala, A. M., Kvaalen, H., Schmidt, A., Johnk, N., Solheim, H., Fossdal, C. G. (2004). Temporal and Spatial Profiles of Chitinase Expression by Norway Spruce in Response to Bark Colonization by Heterobasidion annosum. Appl. Environ. Microbiol. 70: 3948-3953 [Abstract] [Full Text]