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.
2004
Abstract
Introduction: The objectives of the present study were to monitor H. annosum colonization rate (Hietala et al., 2003) and expression of host chitinases in clonal Norway spruce material with differing resistances. Transcript levels of three chitinases, representing classes I, II and IV, were monitored with real-time PCR.Material and MethodsInoculation experiment: Ramets of two 32 -year-old clones differing in resistance were employed as host material. Inoculation and wounding was performed. A rectangular strip containing phloem and cambium, with the inoculation site in the middle, was removed 3, 7 and 14 days after inoculation.Quantification of fungal colonizationMultiplex real-time PCR detection of host and pathogen DNA was performed (Hietala et al., 2003). Quantification of gene expression: Chitinase levels were monitored with Singleplex real-time PCR.Results and ConclusionsThe colonization profiles provided by the quantitative multiplex real-time PCR procedure (Hietala et al., 2003), when combined with spatial and temporal transcript profiling of 3 chitinases, provide a useful basis for identifying defense related genes, and for assessing their impact on pathogen colonization rates.Three days after inoculation, comparable colonization levels were observed in both clones in the area immediately adjacent to inoculation. Fourteen days after infection, pathogen colonization was restricted to the area immediately adjacent to the site of inoculation for the strong clone (589), but had progressed further into the host tissue in the weak (409) clone.Transcript levels of the class II and IV chitinases increased following wounding or inoculation, while the transcript level of the class I chitinase declined following these treatments. Transcript levels of the class II and class IV chitinases were higher in areas immediately adjacent to the inoculation site in 589 than in similar sites in 409 three days after inoculation, suggesting that the clones differ in the rate of chitinase-related signalperception.
2003
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, hereas 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.
Authors
Ari M. Hietala Kari Korhonen R. SenAbstract
Strains of Ceratobasidium bicorne (anamorph uninucleate Rhizoctonia) causing root dieback in nursery-grown conifer seedlings were fruited in the laboratory and the pairing interactions among sibling, single-basidiospore progeny were investigated.No mating reactions were observed. Instead, a high frequency of somatic incompatibility was observed in progeny pairings, indicated by a killing reaction in hyphal anastomosis and by formation of a demarcation line. The F1 progeny could also be fruited, and the level of somatic incompatibility within the F2 progeny remained high, even if lower than in the F1 progeny.The interaction types in pairings within a family of progeny were in all respects similar to those between field isolates, indicating that the species is homothallic. The uninucleate condition of vegetative cells and the basidial characteristics now observed would indicate homokaryotic fruiting, but the possibility of pseudohomothallism remains.We are presently not able to provide an explanation for the mechanism promoting somatic incompatibility in this species, but it seems likely that the classic heterogenic model of somatic incompatibility recognised in basidiomycetes is not applicable here. Alternative mechanisms are discussed.
2002
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 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.
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
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.