Publications
NIBIOs employees contribute to several hundred scientific articles and research reports every year. You can browse or search in our collection which contains references and links to these publications as well as other research and dissemination activities. The collection is continously updated with new and historical material.
2002
Abstract
A quantitative multiplex real-time PCR procedure was developed to monitor the dynamics in Norway spruce (Heterobasidion annosum) pathosystem. The assay reliably detected down to 1 pg of H. annosum DNA and 1 ng of host DNA in multiplex conditions. As a comparative method for quantifying fungal colonization,we applied the ergosterol assay. There was a very high correlation between the results obtained with the two methods, this strengthening the credibility of both assays. The advantages and disadvantages of these assays are discussed.
Abstract
One of our main interests is to learn about the molecular basis of host defense responses, using the coniferous host Norway spruce infected with the pathogen Heterobasidion parviporum as the experimental system. This basidiomycete and the closely related pathogen H. annosum are the major root rot causing pathogens in conifers.To screen host material for differential resistance towards H. parviporum, it is a necessity to quantify the fungal colonization of the host tissues. Therefore, we aimed to develop and compare the sensitivity of a real-time PCR to an ergosterol based method for determining the rate of colonization. We developed a quantitative multiplex real-time PCR procedure that reliably detecting down to 1pg H. parviporum DNA and 1ng host DNA.There was a very high correlation between the fungal-biomass/total-biomass and fungal-DNA/total-DNA rankings obtained with ergosterol and real-time PCR, strengthening the credibility of both methods. The results indicate that this real-time procedure can be a useful method to screen different spruce material for their relative resistance to the pathogen H. parviporum.
Abstract
Determining the level of pathogenic fungi and other microorganisms during colonization of the host is central in phytopathological studies. A direct way to monitor fungal hyphae within the host is microscopic examination, but chitin and ergosterol-levels are commonly used to indirectly measure the amount of fungus present. Recently real-time PCR technology is being used to follow infection agents in host tissues. We study the molecular basis of host defense responses, using the coniferous host Norway spruce infected with the pathogen Heterobasidion parviporum as the experimental system. This basidiomycete and the closely related pathogen H. annosum are the major root rot causing pathogens in conifers. To screen host material for differential resistance towards H. parviporum, it is a necessity to quantify the fungal colonization of the host tissues. Therefore, we aimed to develop and compare the sensitivity of a real-time PCR to an ergosterol based method for determining the rate of colonization, and applied the methods to rank the infection level of the pathogen on the spruce clones 053 and 589. We developed a quantitative multiplex real-time PCR procedure that reliably detecting down to 1pg H. parviporum DNA and 1ng host DNA. There was a very high correlation between the fungal-biomass/total-biomass and fungal-DNA/total-DNA rankings obtained with ergosterol and real-time PCR, strengthening the credibility of both methods. Based on both ergosterol and real-time PCR, it was clear that the clone 053 was hosting more fungal biomass than clone 589. The results indicate that this real-time procedure can be a useful method to screen different spruce material for their relative resistance to the pathogen H. parviporum.
Abstract
Determining the level of pathogenic fungi and other microorganisms during colonization of the host is central in phytopathological studies. A direct way is to monitor fungal hyphae by microscopic examination, but indirect chitin and ergosterol-based assays have been among the most applied methods in determining fungal biomass within host tissues. Recently real-time technology is increasingly receiving attention as a way to follow infection agents in host tissues.We study the molecular basis of host defense responses, using the coniferous host Norway spruce (Picea abies) infected with the basidomycete Heterobasidion annosum as the experimental system. This basidiomycete is the major root rot causing pathogens in conifers of all age classes.In order to screen host material for differential resistance towards H.annosum for both scientific and commercial reasons, it is a necessity to reliably quantify the fungal colonization of the host tissues. Therefore, the aim of this study was to develop and compare the sensitivity of a real-time PCR assay to an ergosterol based method for determining the rate of colonization by H.annosum in inoculated spruce material. We also applied the methods to rank the infection level of the pathogen on the spruce tissue culture clones.We were able to develop a quantitative multiplex real-time PCR procedure that reliably detecting down to 1pg H.annosum DNA and 1ng host DNA in DNA extracted from infected tissues. There was a very high correlation between the fungal-biomass/total-biomass and fungal DNA-total DNA rankings obtained with ergosterol and real-time PCR respectively, strengthening the credibility of both methods.Based on both ergosterol and real-time PCR, it was clear that some spruce clones were faster and more heavily infected than others. These results indicate that both ergosterol and this real-time procedure can be useful methods to screen different spruce material for their relative resistance to the pathogen H.annosum.