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

Abstract

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.Determining the colonization rate of pathogenic fungi and other microbes in host tissues is central to phytopathological studies. One way to monitor fungal hyphae within the host is by microscopic examination of host tissue stained with fungusspecific dyes (15), but this procedure is tedious and difficult to apply in a quantitative manner. Therefore, indirect methods have been developed and applied for estimation of fungal biomass in planta.Ergosterol (6, 14)-based assays commonly have been used to determine fungal biomass within host tissues. Ergosterol is a membrane-associated sterol that occurs almost exclusively in fungi and is degraded in dying mycelium, so it correlates well with markers for metabolic activity (22). Thus, ergosterol assays are commonly used to estimate the amount of living fungal biomass (for examples, see reference 6). In all indirect methods for estimating fungal biomass, a conversion factor (i.e., the relationship between the quantity of the measured component and the total weight of the organism) must be established. The validity of the ergosterol assay for this purpose has been questioned, as the concentration of this component may vary due to nutrient availability and colony age (3). The ergosterol method also lacks specificity and is best suited for laboratory studies, where the presence of multiple fungi can be excluded and will not interfere with the results.PCR-based techniques can be used for sensitive and specific monitoring of phytopathogenic microbes in their natural substrates. Real-time PCR is currently a promising PCR method for quantifying DNA. The measurements are performed during the exponential phase of the reaction, when PCR efficiency is not...

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