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.
2005
Authors
Nina Jøhnk Ari M. Hietala Carl Gunnar Fossdal David B. Collinge Mari-Anne NewmanAbstract
To study the mechanisms of inducible disease resistance in conifers, changes in transcript accumulation in roots of Norway spruce (Picea abies (L.) Karst.) seedlings exposed to the root rot pathogen Ceratobasidium bicorne Erikss. and Ryv. (anamorph: Rhizoctonia sp.) were monitored by differential display (DD). Because C. bicorne attacks root tips, a desiccation treatment was added to exclude genes induced by pathogen-related desiccation stress. The DD analysis was defined by the use of 11 sets of primers, covering about 5% of the transcriptome. A comparison of gene expression in control, desiccation- and pathogen-stressed roots revealed 36 pathogen-induced gene transcripts. Based on database searches, these transcripts were assigned to four groups originating from spruce mRNA (25 transcripts), rRNA (five transcripts), fungal mRNA (two transcripts) and currently unknown cDNAs (four transcripts). Real-time PCR was applied to verify and quantify pathogen-induced changes in transcript accumulation. Of the 18 transcripts tested, nine were verified to be Norway spruce gene transcripts up-regulated from 1.3- to 66-fold in the infected roots. Four germin-like protein isoforms, a peroxidase and a glutathione S-transferase, all implicated in oxidative processes, including the oxidative burst, were predicted from sequence similarity searches. Seven class IV chitinase isoforms implicated in fungal cell wall degradation and a nucleotide binding site-leucine rich repeat (NBS-LRR) disease resistance protein homologue related to pathogen recognition were identified. Several transcript species, such as the NBS-LRR homologue and the germin-like protein homologues, have not previously been identified as pathogen-inducible genes in gymnosperms.
Authors
Nina Jøhnk Ari M. Hietala Carl Gunnar Fossdal Mari-Anne Newman David B. Collinge Harald KvaalenAbstract
Stilbene synthases make the backbone of stilbenes in a single enzymatic step. Many stilbenes are stressinduced antimicrobial phenolics, believed to work in disease resistance. In conifers, stilbenes are found in pine (Pinus), spruce (Picea) and a few other genera.Stilbene synthase isoforms in pine use cinnamyl-CoA to form pinosylvin, these are termed pinosylvin synthases, whereas stilbene synthases in spruce use pcoumaryl- CoA to form resveratrol and are sometimes termed resveratrol synthases.Pinosylvin has been found to be more effective than resveratrol in inhibiting fungal growth and wood decay (Seppnen et al. 2004), and pathogens of non-pinosylvin producing species have been found to be less tolerant of pinosylvin than pine pathogens (Seppnen et al. 2004). In the present study, Norway spruce (Transformation of Norway spruce with the pinosylvin synthase gene, PSS1) was transformed using the biolistic technique with a gene encoding pinosylvin synthase, PSS1, from Scots pine and the E. coli nptII antibiotic resistance gene.Vector constructs carrying PSS1 in sense and antisense, as well as control vectors without PSS1 were transferred into two embryogenic cell lines of Norway spruce, 11703-B63 and 186-3C. Selection condition for transgenic tissue was conferred by nptII in combination with the antibiotic geneticin. Geneticin resistant lines were recovered from all transformation events, a total of 55 lines.NptII was detected by PCR analysis in many of these lines, the majority derived from the cell line 11703 B63. However, nptII protein was detected in just five lines, and several lines of evidence indicate that the transgenic lines obtained in this study might be chimaeras.Fifty-six seedlings were successfully regenerated from antibiotic resistant lines, 50 of these were derived from cell line 11703 B63. All seedlings died during cold storage before further testing could be carried out.
Authors
Carl Gunnar Fossdal Ari M. HietalaAbstract
The root-rot causing fungus Heterobasidion annosum can attack both spruce and pine trees and is the economically most damaging pathogen in northern European forestry. We have monitored the Heterobasidion annosum S-type (fairly recently named H. parviporum) colonization rate and expression of host chitinases and other host transcripts in Norway spruce material with differing resistances. Transcript levels of three chitinases, representing classes I, II and IV, were monitored with real-time PCR. We have also transferred a Class IV chitinase to Arabidopsis as well as its promotor in GFP and YFP reporter constructs. Ramets of two 33 -year-old clones differing in resistance were employed as host material and inoculation and wounding was performed. Multiplex real-time PCR detection of host and pathogen DNA was also performed to follow the colonization of the host tissues by the pathogen and the collapse in host DNA levels in infected regions. Host defense transcript levels, as an indicator of the host defense response, were monitored with singleplex real-time PCR. 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 clone (409). Transcript levels of the class II and IV chitinases increased following wounding or inoculation, while the transcript level of the class Ichitinase 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 pathogen perception and host defense signal transduction. This an earlier experiments using mature spruce clones as substrate indicate that it is the speed of the host response and notmaximum amplitude of the host response that is the most crucial component in an efficient defense in Norway spruce toward pathogenic fungi such as H. annosum.
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.
Abstract
Pathogen colonization and transcript levels of three host chitinases,putatively representing classes I, II, and IV, were monitored with real-time PCR after wounding and bark infection by Heterobasidion annosum in 32-year-old trees of Norway spruce (Picea abies) with low (clone 409) or high (clone 589) resistance to this pathogen. Three days after inoculation, comparable colonization levels were observed in both clones in the area immediately adjacent to inoculation. At 14 days after infection, pathogen colonization was restricted to the area immediately adjacent to the site of inoculation for clone 589 but had progressed further into the host tissue in clone 409. Transcript levels of the class II and IV chitinases increased after wounding or inoculation, but the transcript level of the class I chitinase declined after these treatments. Transcript levels of the class II and class IV chitinases were higher in areas immediately adjacent to the inoculation site in clone 589 than in similar sites in clone 409 3 days after inoculation. This difference was even more pronounced 2 to 6 mm away from the inoculation point, where no infection was yet established, and suggests that the clones differ in the rate of chitinase-related signal perception or transduction. At 14 days after inoculation, these transcript levels were higher in clone 409 than in clone 589, suggesting that the massive upregulation of class II and IV chitinases after the establishment of infection comes too late to reduce or prevent pathogen colonization.
Abstract
We have monitored the H. annosum colonization rate and expression of host chitinases in Norway spruce material with differing resistances. Transcript levels of three chitinases, representing classes I, II and IV, were monitored with real-time PCR. Ramets of two 32 -year-old clones differing in resistance were employed as host material and inoculation and wounding was performed. Quantification of fungal colonization: Multiplex real-time PCR detection of host and pathogen DNA was performed. Chitinase transcript levels were also monitored with real-time PCR. 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 clone (409). 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 signal perception. The spatiotemporal accumulation patterns obtained for the two clones used are consistent with their resistance classifications, these warranting further and more detailed studies on these chitinases.
Authors
Axel Schmidt K. Witzel Gazmend Zeneli D. Martin J. Bohlmann Paal Krokene Trygve Krekling Ari M. Hietala Carl Gunnar Fossdal Erik Christiansen Jonathan GershenzonAbstract
The study of conifer chemical defense has been dominated by investigations of oleoresin and its components. However, the actual function of resin components in plant defense and their mode of action is still uncertain, and the role of other defense compounds is relatively unexplored.We are studying the biochemical and molecular bases of chemical defenses, including terpenes, phenolics and chitinases, in Norway spruce (Picea abies) to learn more about how the accumulation of defense compounds is regulated, with the long-term goal of manipulating defense levels to test their function.Manipulation can be crudely accomplished by treatment with methyl jasmonate, which often mimics the general increases in defenses seen following herbivore or pathogen attack. Such treatment was shown to increase resistance to a fungal associate of bark beetles.To more conclusively test function, isolated genes of defense biosynthetic pathways are being transformed into Norway spruce to produce plants whose defense profiles are altered more precisely.
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
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.