Harald Kvaalen

Temporal and spatial profiles of chitinase expression by Norway spruce in response to bark colonization by Heterobasidion annosum

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.

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.

Genetic variation of wood quality traits and relationships with growth in Picea abies

Abstract Genetic parameters were estimated for wood density and spiral grain in two long-term field trials with families of Picea abies (L.) Karst., and for microfibril angle (MfA) and model-predicted wood stiffness (MOEest) at one of the sites. The trials were located at 600–720 m altitude in Norway and the progenies, which were a sample of 13 half-sib families from plus-trees in a breeding population, were 33 years old from seed when measured. Significant genetic variation (p<0.05) was found for all wood quality traits. The narrow-sense heritability was estimated to be 0.50 for density (across two sites), 0.38 for MfA, 0.29 for MOEest and 0.37 for spiral grain (across two sites). No significant genotype by environment interactions were found for density or spiral grain (p>0.05). Genetic relationships between ring width and wood quality traits were negative for density and MOEest, and positive for MfA. Site index and competition had major effects on wood density and predicted MOEest but not on MfA and spiral grain.

Spatial patterns in hyphal growth and substrate exploitation within Norway spruce stems colonized by the pathogenic white-rot fungus Heterobasidion parviporum.

ABSTRACT In Norway spruce, a fungistatic reaction zone with a high pH and enrichment of phenolics is formed in the sapwood facing heartwood colonized by the white-rot fungus Heterobasidion parviporum. Fungal penetration of the reaction zone eventually results in expansion of this xylem defense. To obtain information about mechanisms operating upon heartwood and reaction zone colonization by the pathogen, hyphal growth and wood degradation were investigated using real-time PCR, microscopy, and comparative wood density analysis of naturally colonized trees with extensive stem decay. The hyphae associated with delignified wood at stump level were devoid of any extracellular matrix, whereas incipient decay at the top of decay columns was characterized by a carbohydrate-rich hyphal sheath attaching hyphae to tracheid walls. The amount of pathogen DNA peaked in aniline wood, a narrow darkened tissue at the colony border apparently representing a compromised region of the reaction zone. Vigorous production of pathogen conidiophores occurred in this region. Colonization of aniline wood was characterized by hyphal growth within polyphenolic lumen deposits in tracheids and rays, and the hyphae were fully encased in a carbohydrate-rich extracellular matrix. Together, these data indicate that the interaction of the fungus with the reaction zone involves a local concentration of fungal biomass that forms an efficient translocation channel for nutrients. Finally, the enhanced production of the hyphal sheath may be instrumental in lateral expansion of the decay column beyond the reaction zone boundary.

Indications of heightened constitutive or primed host response affecting the lignin pathway transcripts and phenolics in mature Norway spruce clones

Two mature clones of Norway spruce (Picea abies (L.) Karst.) that have previously been shown to have differential degrees of resistance towards the necrotrophic pathogen Heterobasidion parviporum (Niemelä & Korhonen) were compared with respect to the primed defense expression of transcripts related to biosynthesis of lignin, stilbenes and other phenolic compounds from one year to the next. The hosts response to physical wounding and pathogen inoculation was examined in the initial year, whereas indications of heightened basal defense level or primed response, and responses to re-wounding, were examined the following year. The responses of the two clones to wounding and pathogen inoculation, examined in the initial year, differed; the increases in lignin and phenolics were more distinct in response to the pathogen than to wounding alone. The more resistant clone 589 had higher initial lignin concentrations in the cell walls when compared with clone 409, and these remained higher in clone 589 over both years and increased after the treatments. Both clones responded at the transcriptional and chemical levels to wounding; changes were evident both in the initial wounds and when re-wounded the following year. There were distinct differences in the basal transcript levels of the lignin pathway-related genes, phenolics and total lignin levels in healthy tissue from the initial year to the following year indicative of a primed host response or at least altered constitutive level of defense expression.

Local and systemic changes in expression of resistance genes, nb-lrr genes and their putative microRNAs in norway spruce after wounding and inoculation with the pathogen ceratocystis polonica

BackgroundNB-LRR resistance proteins are involved in recognizing pathogens and other exogenous stressors in plants. Resistance proteins are the first step in induced defence responses and a better understanding of their regulation is important to understand the mechanisms of plant defence. Much of the post-transcriptional regulation in plants is controlled by microRNAs (miRNA). We examined the expression of five Norway spruce miRNA that may regulate NB-LRR related transcripts in secondary phloem (bark) of resistant Norway spruce after wounding and inoculation with the necrotrophic blue stain fungus Ceratocystis polonica.ResultsThe plants of this clone recovered from both the pathogen inoculations and wounding alone. We found local and systemic induction of the resistance marker genes PaChi4, PaPAL and PaPX3 indicative of an effective induced host defence response. There were minor local and systemic changes in the expression of five miRNAs and 21 NB-LRRs between healthy and treated plants. Only five putative NB-LRRs (PaLRR1, PaLRR3, PaLRR14, PaLRR15 and PaLRR16) showed significant increases greater than two-fold as a local response to C. polonica. Of all NB-LRRs only PaLRR3, the most highly differentially regulated NB-LRR, showed a significant increase also due to wounding. The five miRNAs showed indications of an initial local and systemic down-regulation at day 1, followed by a later increase up to and beyond the constitutive levels at day 6. However, the initial down-regulation was significant only for miR3693 and miR3705.ConclusionsOverall, local and systemic expression changes were evident only for the established resistance marker genes and PaLRR3. The minor expression changes observed both for the followed miRNAs and their predicted NB-LRR targets suggest that the expression of most NB-LRR genes are maintained close to their constitutive levels in stressed and healthy Norway spruce plants.

The genetic and economic gains from forest tree breeding programmes in Scandinavia and Finland

ABSTRACT Plantations of genetically improved forest trees are critical for economic sustainability in forestry. This review summarizes gains in objective traits and the resulting economic impact of tree breeding programmes in Scandinavia and Finland. Genetic improvement of forest trees in these countries began in the late 1940s, when the first phenotypically superior plus-trees were selected from natural environments. The main findings from this review are that (i) tree breeding can increase volume growth in the range 10–25%, and (ii) the bare land value associated with genetically improved trees gives a better return on investment and a shorter rotation period compared to the unimproved forests. As some Nordic countries are quite dependent on the forest industry, breeding programmes that have resulted in economic gains have been beneficial for society. Growth and wood quality traits are often adversely correlated, and the weighting of traits from an economic perspective could provide an index for determining maximum profit from breeding. Tree breeding faces an array of challenges in the future, such as changes in silviculture, climate, new pests and diseases, and demand for wood-based products.

The Pathogenic White-Rot Fungus Heterobasidion parviporum Responds to Spruce Xylem Defense by Enhanced Production of Oxalic Acid

Pathogen challenge of tree sapwood induces the formation of reaction zones with antimicrobial properties such as elevated pH and cation content. Many fungi lower substrate pH by secreting oxalic acid, its conjugate base oxalate being a reductant as well as a chelating agent for cations. To examine the role of oxalic acid in pathogenicity of white-rot fungi, we conducted spatial quantification of oxalate, transcript levels of related fungal genes, and element concentrations in heartwood of Norway spruce challenged naturally by Heterobasidion parviporum. In the pathogen-compromised reaction zone, upregulation of an oxaloacetase gene generating oxalic acid coincided with oxalate and cation accumulation and presence of calcium oxalate crystals. The colonized inner heartwood showed trace amounts of oxalate. Moreover, fungal exposure to the reaction zone under laboratory conditions induced oxaloacetase and oxalate accumulation, whereas heartwood induced a decarboxylase gene involved in degradation of oxalate. The excess level of cations in defense xylem inactivates pathogen-secreted oxalate through precipitation and, presumably, only after cation neutralization can oxalic acid participate in lignocellulose degradation. This necessitates enhanced production of oxalic acid by H. parviporum. This study is the first to determine the true influence of white-rot fungi on oxalate crystal formation in tree xylem.

Xylem defense wood of Norway spruce compromised by the pathogenic white-rot fungus Heterobasidion parviporum shows a prolonged period of selective decay.

Heterobasidion parviporum, a common pathogenic white-rot fungus in managed Norway spruce forests in northern and central Europe, causes extensive decay columns within stem heartwood of the host tree. Infected trees combat the lateral spread of decay by bordering the heartwood with a fungistatic reaction zone characterized by elevated pH and phenol content. To examine the mode of fungal feeding in the reaction zone of mature Norway spruce trees naturally infected by H. parviporum, we conducted spatial profiling of pectin and hemicellulose composition, and established transcript levels of candidate fungal genes encoding enzymes involved in degradation of the different cell wall components of wood. Colonized inner heartwood showed pectin and hemicellulose concentrations similar to those of healthy heartwood, whereas the carbohydrate profiles of compromised reaction zone, irrespective of the age of fungal activity in the tissue, indicated selective fungal utilization of galacturonic acid, arabinose, xylose and mannose. These data show that the rate of wood decay in the reaction zone is slow. While the up-regulation of genes encoding pectinases and hemicellulases preceded that of the endoglucanase gene during an early phase of fungal interaction with xylem defense, the manganese peroxidase gene showed similar transcript levels during different phases of wood colonization. It seems plausible that the reaction zone components of Norway spruce interfere with both lignin degradation and the associated co-hydrolysis of hemicelluloses and pectin, resulting in a prolonged phase of selective decay.

A high-throughput X-ray-based method for measurements of relative wood density from unprepared increment cores from Picea abies

We have described a high-throughput method for ring width and relative wood density analysis of unprepared increment cores based on conventional computer tomography scanners (CT scanners) used in health care. The method was developed for analysis of 5-mm increment cores collected in forest genetic and tree breeding trials. For these studies, the relative differences between individuals are more important than the exact value. The method is highly efficient in terms of quantitative data collection from large number of samples. There are, however, limitations in terms of resolution and accuracy on very small volumes of wood that must be considered when using the method to measure individual growth rings or increment cores thinner than 5 mm. However, in studies comprising large number of samples, the use of cost-efficient CT scanning on the major part of the material, combined with more laborious high-resolution scanning on sub-samples can be a good combination for efficient determination of radial variation in growth rate and wood density.