Harald Kvaalen

Research Professor

(+47) 469 51 314
harald.kvaalen@nibio.no

Place
Ås H8

Visiting address
Høgskoleveien 8, 1433 Ås

Abstract

Several studies have shown the positive effect of nitrogen fertilization on conifer growth. In young Norway spruce (Picea abies) stands, an additional effect of including a mixture of other nutrients has often, but not always, been found. We studied effects of repeated fertilization in 28 stands with young Norway spruce in central Norway. The treatments consisted of plots without nutrient addition (Control), fertilization with 150 kg N ha−1 (150 N), and fertilization with 150 kg N plus addition of P, K, Mg, B, Mn and Cu (150 N + mix), repeated three times with approximately eight years interval. There was a clear positive effect on volume increment of the 150 N and 150 N + mix treatments compared to Control, and the effect was significantly higher for 150 N + mix than for 150 N. Fertilization had a stronger effect in the first fertilization period than in the second, while the third period was intermediate. The effect of 150 N + mix was strongest at plots > 300 m a.s.l. However, this correlation may be due to geological conditions rather than elevation. Further studies are needed to find out under which edaphic conditions a nutrient mixture will increase growth substantially in young spruce stands.

Abstract

In the Nordic-Baltic region, there has been a growing concern about an increasing occurrence of multiple tops in young stands of Norway spruce. There is however a lack of documentation on the amount of such damages, and the causal agents involved. In two separate studies in SE Norway, we assessed the frequency of multiple tops in young sapling-sized stands, and studied the relationship between the occurrence of multiple tops and lammas growth the previous growing season on the sample trees. Study 1 included 44 planted and 10 naturally regenerated stands, while Study 2 included 68 planted stands with information on seed source. Among sample trees with multiple tops, 57% (Study 1) and 32% (Study 2) had signs of lammas growth the previous autumn, either in the form of an extended leading shoot or swollen bud. Site index as well as sample tree height were positively correlated to the occurrence of both lammas growth and multiple tops in both studies. In Study 1 we show that the probability of lammas growth was significantly higher in planted than in naturally regenerated stands. In Study 2 we show that it was higher in stands planted with seedlings grown from stand-origin seeds compared with improved seed material. Furthermore, the results of both studies show that lammas growth occurs most frequently among the dominant trees in the stand.

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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.

Abstract

Globally, billions of tons of carbon sequestered in trees are annually recycled back to the atmosphere through wood decomposition by microbes. In Norway, every fifth Norway spruce shows at final harvest infection by pathogenic white-rot fungi in the genera Heterobasidion and Armillaria. As these fungi can mineralize all components of wood, we predicted that they have a significant carbon footprint. Gas samples taken from infected stems were analyzed for CO2 and CH4 concentrations, and wood samples from different parts of the decay columns were incubated under hypoxic (4% O2) and anoxic laboratory conditions. In spring and summer the stem concentrations of CO2 were generally two times higher in trees with heartwood decay than in healthy trees. For most of the healthy trees and trees with heartwood decay, mean stem concentrations of CH4 were comparable to ambient air, and only some Armillaria infected trees showed moderately elevated CH4. Consistently, low CH4 production potentials were recorded in the laboratory experiment. Up-scaling of CO2 efflux due to wood decay in living trees suggests that the balance between carbon sequestration and emission may be substantially influenced in stands with high frequency of advanced root and stem heartwood decay.

Abstract

We compared gene expression in Norway spruce secondary phloem (bark) and developing xylem (sapwood) in response to the necrotrophic pathogen Heterobasidion parviporum, wounding and methyl jasmonate (MeJ). The pathogen induced systemic and local up-regulation of PaPX3, PaPX2 and PaChi4 in both bark and sapwood that returned to constitutive levels as the plants recovered from the infection, whereas the local responses to MeJ were similar in both tissues but was longer lasting for PaPX3 and PaChi4. Genes involved in lignin biosynthesis (PaPAL1, PaPAL2, PaC4H3/5 and PaHCT1) were up-regulated locally in the bark in response to pathogen and wounding whereas MeJ induced a similar but stronger local response. The ethylene biosynthesis related transcripts PaACO and PaACS did not increase in response to MeJ treatment or the pathogen, however it increased both locally and systemically as a response to wounding in the sapwood. These results demonstrate that the local and systemic host responses to pathogen infection and wounding largely correspond and reveal striking similarities between the local response to a necrotroph, wounding and MeJ treatment in both bark and living wood.

Abstract

Background: NB-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. Results: The 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. Conclusions: Overall, 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.

Abstract

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.

Abstract

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 proWling of pectin and hemicellulose composition, and established transcript levels of candidate fungal genes encoding enzymes involved in degradation of the diVerent cell wall components of wood. Colonized inner heartwood showed pectin and hemicellulose concentrations similar to those of healthy heartwood, whereas the carbohydrate proWles 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 diVerent 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.

Abstract

We have recently found that Norway spruce (Picea abies (L.) Karst.) can rapidly adjust its adaptive performance, probably through an epigenetic mechanism. This appears to employ a kind of long-term memory of temperature sum and (probably) photoperiod from the time of its embryo development. In our research we made identical controlled crosses and produced seed lots under controlled temperature and day-length conditions and later observed phenology, growth and hardiness traits in the progenies. It was repeatedly found that temperature conditions during seed set, in particular, influence the phenotypes of the offspring; seedlings from seeds produced under warm conditions have later terminal bud set and reduced autumn frost hardiness than those from seed produced under colder conditions, and thus perform like a more southern provenance. When embryonic clones were derived from mature zygotic embryos and were cultured at different temperatures, the plants cultured under warm in vitro temperature were the last to set bud and grew taller than those cultured at lower temperatures. Progenies produced in Norway by Central European mother trees had a bud set curve skewed towards that of the local Norwegian performance. A comparison of the performance of seedlings from seeds collected in the same provenance regions in 1970 and 2006 shows that the more recent seed lots consistently produce taller seedlings with a later bud set, probably due to higher temperatures during seed production in 2006. The effect of reproductive environment has been shown to persist for years. It mimics the variation between provenances from different latitudes and altitudes and may explain much of the observed variability in bud set and early height growth between natural populations of Norway spruce. The observed phenomenon suggests an epigenetic mechanism in the developing embryo, either zygotic or somatic, that senses environmental signals such as temperature and influences adaptive traits. Research is underway to understand the molecular basis of this mechanism. We will discuss the implications of this epigenetic phenomenon for the interpretation of provenance differences, for tree breeding and for its possible role in adaptation to climate change.

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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 (p0.05) 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 (p0.05). 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.

Abstract

Clonal variation towards resistance has been observed in Norway spruce Heterobasidion annosum s.l. (H.a). H.a. is the main cause of root rot and has a severe economic impact on an economically important conifer tree species. Annual financial losses are in the hundreds of millions of Euros annually. Less susceptible clones appear to have an efficient system of recognizing the pathogen and initiating early defense signalling events. Active defense responses can be started locally and transmitted systemically. This work focus on the expression both spatially (systemically) and temporally in this pathosystem. Two-year-old, somatic saplings of the Norway spruce clone were challenged with H.a., wounded, methyl jasmonate painted and compared to untreated controls and ninety plants were used for the experiment. Stem samples were collected at 1, 3, 6 and 13 days post inoculation (d.p.i). The stem of the saplings were divided into sections along its length and the bark and wood separated from each other at time of collection. In order to see local response an area of 1cm including the site of inoculation was collected, while the spatial (systemic) response was assessed in sections collected at distances of 3 and 6cm away from the site of inoculation. The separated bark and wood were analysed for differential gene expression by qRT-PCR, and the results from peroxidases (PaPX3 and PaPX2) and a chitinase (PaChi4) are presented. Both local and systemic up- and down-regulation were observed at the transcriptional level in both bark and wood, up to 2000 fold local increase in expression was observed for PaChi4.

Abstract

MicroRNAs (miRNAs) are endogenous small RNAs that can have large-scale regulatory effect and could participate in epigenetic regulation of gene expression in plants. We show for the first time that temperature during zygotic embryogenesis and seed maturation in Norway spruce regulates an “epigenetic memory” in the progeny, regenerated through somatic embryogenesis. The warmer the in vitro temperature applied, the later the regenerated plants formed terminal buds in the common environment the second growth season. The differences were very large, and similar in size to a provenance separation of 4 – 6 degrees of latitude (Kvaalen and Johnsen, 2008). To study a molecular mechanisms of a memory from embryo development we have prepared two concatemerized small RNA libraries representing small RNAs predominantly expressed in plants growing from seeds obtained after embryogenesis in cold environment (CEL) and warm environment (WEL) after short day (SD) treatment (going to bud set). In total we obtained 201 different small RNAs, with dominated length of 21-nt, 123 from WEL and 93 from CEL. Using multiple methods, including BLAST, sequence alignment and sequence folding we found 27 novel candidate miRNAs and only 3 earlier described. Additionally 103 small RNAs have exact matches in spruce EST database, which could be their putative targets and 67 small RNAs have no matches. We used quantitative RT–PCR to study the expression patterns of 31 chosen candidate miRNAs and monitor the occurrence stage-dependent miRNA-mediated cleavage for 4 regions of putative mRNA targets. All miRNA show difference in transcript levels after SD treatment and 12 miRNAs show constitutively differential expression in progeny from CE and WE. Kvaalen H, Johnsen O (2008) Timing of bud set in Picea abies is regulated by a memory of temperature during zygotic and somatic embryogenesis. New Phytologist 177: 49-59

Abstract

Two mature clones of Norway spruce (Picea abies (L.) Karst.) shown to have different level of resistance towards inoculation of Heterobasidion parviporum were compared with respect to spatiotemporal expression of transcripts related to biosynthesis of lignin, stilbenes and other phenolic compounds in response to fungal inoculation and physical wounding. Both clones responded to H. parviporum and physical wounding at transcriptional and chemical levels. Taxifolin, detected in the resistant clone only, increased in concentration following both wounding and inoculation. Concentrations of stilbenoid glucosides were highest in the susceptible clone. Following wounding or inoculation, concentrations of these glucosides increased in the susceptible clone, and quantities of their corresponding aglycones increased dramatically in both clones close to the treatment point. Significant changes in transcription were detected over the entire lesion length for all transcripts, and only the changes in a few transcripts indicated a response to inoculation with H. parviporum differing from that caused by wounding alone. The resistant clone had higher basal concentrations of lignin (LTGA) compared to the susceptible clone; concentrations increased in both clones after wounding and wounding plus inoculation treatments, but remained consistently higher in the resistant clone, suggesting higher lignin levels in the cell walls compared to the susceptible clone. In addition, the transcript level in the same clones was also measured the following year and we saw indications of primed defences for a number of gene products likely resulting from the inoculations performed 12 months prior.

Abstract

In forest trees, environmental conditions during the reproduction can greatly influence progeny performance. We here suggest that temperature during zygotic embryogenesis and seed maturation regulates an “epigenetic memory” in the progeny, involving differential expression of genes that may regulate bud phenology, cold acclimation and embryogenesis in Norway spruce. Conditions colder than normal advance the timing whilst temperatures above normal delay the onset of these processes. The altered performance lasts for many years. The seedlings actually remember the temperatures and photoperiod prevailing during zygotic embryogenesis and seed maturation. We show for the first time that regenerated plants, cloned through somatic embryogenesis, express a memory of the temperatures applied during embryo development whilst growing in a common greenhouse environment. The warmer the in vitro temperature applied, the later the regenerated plants formed terminal buds in the common environment the second growth season. The differences were very large, and similar in size to a provenance separation of 4 – 6 degrees of latitude. To study a molecular mechanisms of a memory from embryo development identical crosses were performed, seed were produced in different temperature regimes (cold and warm embryonic history) and seedling were grown under long day (LD = continuous light) and short day (SD = 12 h night) conditions. We have prepared two subtracted cDNA libraries, forward and revers, representing genes predominantly expressed in plants growing from seeds obtained after embryogenesis in cold environment (CE) and warm environment (WE) after short day treatment (going to bud set). Annotation reveals considerable differences in studied transcriptomes. More than 50% of contigs were unknown. So obtained subtracted libraries is a good source of candidate genes. We used quantitative RT–PCR to study the expression patterns of 34 chosen candidate genes. Just two putative genes among them with no matching in the Database and one reverse transcriptase show constitutively differential expression in progeny from CE and WE

Abstract

It has been shown previously that height growth and bud phenology are influenced by the temperature during zygotic embryogenesis in Picea abies.To test whether this phenomenon operates within individual plants, clones produced through somatic embryogenesis were used. Seeds were from a full-sib family produced in both a cold (outdoor) and a warm (inside a glasshouse) environment. Embryogenic clones derived from mature zygotic embryos from both crossing environments were cultured at 18, 23 and 28 during the proliferation and embryo maturation steps.After the second growing season in a glasshouse, plants from the warm seed production environment were taller and had significantly later bud set. For the first time, it is also shown that plants are influenced by the in vitro temperature during somatic embryo development. The warmer the temperature, the later the plants formed terminal buds. The differences were similar to those produced by a provenance separation of 4-6 degrees of latitude.The results indicate that there exists a mechanism in P. abies that operates during embryo development and adjusts the timing of bud set in accordance with the temperature conditions in which the mother tree lives. This in turn counteracts negative effects of gene flow among populations located along altitudinal and latitudinal gradients.

Abstract

Norway spruce (Picea abies (L.) Karst.) has a natural distribution in the northern parts of Europe and Asia and is economically the most important tree species grown in the Nordic countries. A common threat to Norway spruce is the basidiomyceteous fungus Heterobasidion parviporum Niemelä and Korhonen. H. parviporum mainly attacks Norway spruce, although Siberian fir (Abies sibirica Ledeb.) and Scots pine (Pinus sylvestris L.) occasionally get infected. One obstacle to studying host/pathogen interaction in conifers has been the limited availability of mature clones for controlled inoculations, as genetic variation within the host material and the lack of replicates complicate interpretation of the results. Somatic embryogenesis, rooted cuttings, and tissue cultures may provide solutions for this problem. Tissue cultures from mature Norway spruce trees have been proposed as a possible model system for assessing resistance toward fungal pathogens. Recent data on chitinase isoform activity in the Norway spruce/H. parviporum pathosystem are encouraging; clonal variation was observed in the isoforms affected by inoculation, and the isoforms showing increased band intensity following bark inoculation by H. parviporum were also induced in the inoculated tissue cultures of the corresponding clones. To investigate the biological relevance of tissue cultures in host-pathogen interaction studies, transcript levels of selected host and pathogen genes in tissue cultures of Norway spruce were compared to those in bark of 33-year-old ramets of the same clones upon challenge by the pathogenic fungus H. parviporum. Similar transcript profiles of the pathogen and host genes were observed in both tissues, this supporting the use of tissue cultures as experimental material for the pathosystem. Higher transcript levels of the host genes phenylalanine ammonia lyase, peroxidase, and glutathione-S-transferase were observed in the more resistant clone #589 than in the less resistant clone #409 during the early stages of colonization. The most striking difference between the spruce clones was related to gene transcript levels of a class IV chitinase, which showed a continuous increase in clone #409 over the experimental period, with a possible association of this gene product to programmed cell death. Several of the fungal genes assayed were differentially expressed during colonization, including putative glutathione-S-transferases, laccase, cellulase, cytochrome P450 and superoxide dismutase genes. The transcriptional responses suggest an important role for the antioxidant systems of both organisms.

Abstract

In field experiments, clones of Norway spruce [Picea abies (L.) Karst.] showed different degrees of resistance against pathogenic fungi inoculated into the bark that correlate with differences in polyphenolic parenchyma (PP) cells of the bark. Cells of spruce callus cultures, particularly towards the callus surface, resemble PP cells and this study looks at changes in callus cells during infection and the relative resistance of cultures from clones of low (weak) or high (strong) resistance to fungal infection. Callus cultures, initiated from trees with different resistance, were co-inoculated with Ceratocystis polonica (Siem.) C. Moreau and Heterobasidion annosum (Fr.) Bref. Callus cells from strong clones resemble PP cells of bark tissue from strong clones, having more polyphenolic bodies, while callus cells from weak clones are more similar to PP cells from those clones, which have less extensive phenolic bodies. Callus cultures from trees with weak resistance were more quickly overgrown by both species of pathogenic fungi than cultures from trees with strong resistance. Callus cells of infected cultures showed changes similar to activated PP cells of bark, including enhanced accumulation of polyphenolics. Phenolic bodies were more numerous and more extensive (larger and denser) in callus cells of strong versus weak clones under all conditions. Thus, callus cells may perform similar functions in defense as PP cells in the bark. Callus from trees of varying resistance seem to reflect the relative resistance of the trees from which they are derived, and this study indicates that some mechanisms of resistance can be studied using callus from trees of different resistance.

Abstract

Rhizoctonia solani was frequently isolated in the Italian Alps from ursery-grown European beech (Fagus sylvatica) seedlings displaying symptoms of cotyledon rot. Koch?s postulates were verified and mode of infection of the associated isolates was investigated with light and scanning electron microscopy. Population structure of the pathogen was investigated by scoring the anastomosis reaction type in pairings between different isolates from the same seedbed. One pathogen genotype showed a large distribution area within the seedbed, this implying that the inoculum had been building up in the seedbed over a longer time period. Hyphal anastomosis tests and sequence analysis of the internal transcribed spacer (ITS) region of ribosomal DNA indicated that the pathogen belongs to AG 2-1 of R. solani. ITS sequence analysis indicates that the isolates from beech are closely related to R. solani isolates causing a disease on tulip. The striking similarities between the two diseases are discussed.

Abstract

Seeds of Abies lasiocarpa (Hook.) Nutt. (subalpine fir) were dissected, and the different parts were analyzed for elemental composition. The data were used to design a novel growth medium for initiation of somatic embryogenesis. Embryogenic cultures were initiated from immature zygotic embryos from six open-pollinated families of A. lasiocarpa on three different media.The frequency of initiation was the highest in early to mid-July when the zygotic embryo explants were ca. 0.8 mm long. Thereafter the response declined rapidly.The culture media did not significantly affect the initiation frequencies, but the subsequent growth and culture survival was dependent on the culture medium. On the Schenk Hildebrandt medium, many cultures ceased to grow and died. Several of the decaying cultures were rescued after transfer to one of the new media. Proliferating cultures could be stimulated to produce mature embryos. Of 2510 mature somatic embryos, 212 (8.4%) converted to plants, and 35 plants have grown over two periods.

Abstract

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.

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

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.

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

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.

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

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.

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

The Norwegian intensive monitoring programme of forest condition has recorded rainfall, throughfall and soil water data from 1986 at 16 forest plots. Using covariance analysis, this study has examined short term and episodic influences on soil water ionic concentration at three of the plots, and identified both seasonal and long-term temporal trends. Acidity has decreased in bulk precipitation and throughfall, and the concentrations of base cations in the organic soil horizon have increased. Nevertheless, there is evidence of continued acidification in the organic and mineral soil horizons, though of a small scale. The influence of sea salt and drought effects on soil water chemistry are examined, but found to be unimportant in causing acidification effects such as increased soil aluminium concentration.

Abstract

Genetic associations between initiation of embryogenic tissue (ET) and susceptibility to the phytopathogenic fungi Ceratocystis polonica (Siem.) C. Moreau and Heterobasidion annosum (Fr.) Bref. in Norway spruce have been studied by initiating ET from zygotic embryos of mature seeds collected from 19 clones tested for susceptibility to the pathogens in a clonal field trial.Initiation frequencies varied significantly among clones (families), ranging from 12 to 56%. The family variance component accounted for more than 40% of the total variance in initiation frequency of ET. The estimates of broad-sense heritability of fungus susceptibility of the clones ranged from 0.12 for length of phloem necrosis after low-density inoculation with H. annosum to 0.55 for blue-stained sapwood after mass inoculation with C. polonica.None of the susceptibility measures showed any phenotypic correlation with initiation of embryogenic tissue. Genetic correlations and their standard errors were estimated by bootstrapping. Two measures of fungal susceptibility correlated genetically with initiation of ET; estimated at 0.58 for lesion length after inoculation with C. polonica and 0.29 for H. annosum lesion length. A better measure of susceptibility, blue-stained sapwood following inoculation with C. polonica, was not correlated with initiation of ET.

Abstract

We have studied how callus cultures from two clones of Norway spruce influence the growth of two pathogens, Ceratocystis polonica and Heterobasidion annosum, when co-cultivated in vitro. In field experiments, trees of clone 409 were susceptible to both fungi, whereas clone 589 was less affected. Callus was cultured on medium containing cytokinins (benzylaminopurine, kinetin) and with or without auxin (2,4-dichlorphenoxy acetic acid). For co-cultivation with fungus, one piece of callus was placed towards the edge of each Petri dish. One and 14 days after inoculation with callus the dishes were co-inoculated with the fungus. Both clones strongly stimulated the initial growth of both fungi. Clone 589 inhibited the growth of both fungi when the fungi were inoculated one day after the callus. When the callus was cultured on medium without auxin for 14 days before co-inoculation clone 589 strongly inhibited the growth of both fungi, whereas clone 409 inhibited H. annosum only.

Abstract

The influence of light quality on the proliferation of embryogenic tissue of three genotypes of Norway spruce (Picea abies [L.] Karst), with different capacities for mature somatic embryo production, was studied.The proliferating tissues were subjected to light from commercially available light sources: Philips TLD Warm White 36W/29, Philips TLD Blue 18W/18, Philips TLD Red 36W/15, Osram L Fluora 36W/77 and Sylvania Far Red 7080, for 18 h a day with the photon flux (PAR) at 30 mol m-2 s-1.The effect of light quality on the growth of embryogenic tissue was strongly genotype dependent. In genotype 164-4 tissue proliferation was strongly inhibited by blue and red light. Genotype 86:52 reacted in a similar way, but not as strongly as 164-4, whereas the tissue of genotype 186-3 was almost insensitive to light quality and grew fast in all light conditions.

Abstract

An efficient Biolistic transformation technology was developed to stably transform Picea abies (L.) Karst. Several embryogenic tissue lines were tested for proliferation on standard embryogenesis media. Transient transformation studies with these lines were performed to optimize the parameters for genetic transformation. Selection conditions for transgenic tissue based on the nptII resistance gene in combination with the antibiotic geneticin were defined such that only transgenic P. abies lines were able to develop. Nontransgenic tissue was completely inhibited under these conditions. Stable integration of a uidA reporter gene and a nptII resistance gene into the genome of P. abies was achieved and more than 200 mature embryos were regenerated for every transformation event. Histochemical and fluorometric analysis indicated strong expression of the uidA gene in transgenic material. ELISA studies to detect and quantify the nptII gene product as well as polymerase chain reaction and Southern blotting confirmed the presence and integration of uidA and nptII genes into the P. abies genome. Transgenic P. abies plants from nine independent transformation events were recovered and are currently growing in a greenhouse for genetically modified organisms, awaiting field release.

Abstract

We studied how light from different light sources influences germination and postgerninative growth of plants from somatic embryos and seeds of Norway spruce (Picea abies [L.] Karst).Somatic embryos of three spruce genotypes and seeds were subjected to light from commercially available light sources: Philips TLD Blue 18W/18 (BL), Osram Fluora (FL), Philips Cool White TL 50W/33 (CW), Osram Warm White 18W/30 (WW), Philips Yellow 36W/16 (YE) and Philips TLD Red 36W/15 (RE), 18 h a day, with a photon flux (PAR) at 30 mu mol m(-2) s(-1). After 6 weeks the germination frequencies of the somatic embryo-derived plantlets were 50% under BL and 98% under RE. The corresponding mean root lengths were 6.7 and 15.4 mm. In somatic embryo-derived plantlets cultured under BL, FL, CW and WW, both roots and hypocotyls turned brown, presumably due to production of phenolic substances. Browning was less severe in somatic embryo-derived plantlets cultured under RE and YE. Under RE, the epicotyl elongated in 37% of the plantlets after 6 weeks, compared with 70% under the other light sources. Seed germination and postgerminative seedling growth was modestly influenced by light from these light sources. RE and WW initially delayed germination as compared with BL, FL and CW, but after 2 weeks, more than 90% of the seeds had germinated under all light sources. In conclusion, germination and postgerminative growth of somatic embryos of spruce is sensitive to differences in light quality, whereas seed germination and seedling growth is not.

Abstract

Ethylene production from an embryogenic culture of Norway spruce (Picea abies L.) was generally low, ca 2.5 nl g(-1) h(-1), whereas 1-aminocycloprapane-1-carboxylic acid (ACC) concentration was high, fluctuating between 50 and 500 nmol g(-1) during the 11-day incubation period. Hypoxia (2.5 and 5 kPa O-2) rapidly inhibited ethylene production without subsequent accumulation of ACC. Exogenous ACC (1, 10 and 100 mu M) did not increase ethylene production, but the highest concentrations inhibited tissue growth. Ethylene (7 mu l l(-1)) did not inhibit growth either when supplied as ethephon in the medium or in a continuous flow system. Benzyladenine (BA) had little effect on ethylene production, although it was necessary for sustaining the ACC level. Omission of 2,4-dichloro-phenoxyacetic acid (2,4-D) from the medium caused ethylene production to increase from about 2.5 to 7 nl g(-1) h(-1) within the 11-day incubation period. Although 2,4-D did not specifically alter the endogenous level of ACC, the lowest ACC level, 33 nmol g(-1), was observed in tissue treated with 2,4-D (22.5 mu M) and no BA for 11 days. Data from this treatment were used to estimate the kinetic constants for ACC oxidase, the apparent K-m was 50 mu M and V-max 2.7 nl g(-1) h(-1). Growth of the tissue was strongly inhibited by 2,4-D in the absence of BA, but weakly in the presence of BA (4.4 mu M). The results suggest that ethylene or ACC may be involved in the induction of embryogenic tissue and in the early stages of embryo maturation.

Abstract

It is known that reducing the partial pressure of O2 influences the induction of somatic embryogenesis. We tested the hypothesis that O2 causes changes in the endogenous levels of exogenously supplied benzyladenine (BA) or 2,4-dichlorophenoxyacetic acid (2,4-D).Embryogenic tissue of Picea abies was incubated under reduced (2.5, 5 kPa) and ambient (21 kPa) levels of O2 for 1, 3, 7 and 11 days and the endogenous concentrations of BA and 2,4-D were measured. For all treatments the concentration of BA in the tissue increased until the third day. At day 3, the ratio of BA in the tissue relative to the initial concentration in the medium, was 3.9, 2.8 and 1.9 for tissue incubated under 2.5, 5 and 21 kPa O2, respectively. The BA concentration then declined gradually. Uptake of 2,4-D was inhibited at low O2 levels.However, 2,4-D gradually accumulated in tissue grown under hypoxia, so that high levels were reached by day 11. These shifts in the BA and 2,4-D levels also caused a transient increase in the BA to 2,4-D ratio in tissue incubated under hypoxia. Although relevant for the previously reported effects of oxygen on induction of embryogenic tissue, it is unlikely that oxygen-induced alterations in BA and 2,4-D levels alone suffice to explain these findings.

Abstract

Embryogenic cultures of red spruce (Picea rubens Sarg.) and Norway spruce (Picea abies (L.) Karst.) were initiated from dissected mature zygotic embryos. The tissues were grown on either proliferation medium or maturation medium. On proliferation medium, the embryogenic tissue continued to produce early stage somatic embryos (organized meristems attached to elongated, suspensor-like cells), whereas on maturation medium fully mature embryos developed from the embryonic tissue. Analysis of polyamines in tissues grown on these two media showed that: both putrescine and spermidine concentrations were always higher in cultures grown on proliferation medium than in cultures grown on maturation mediumin both species, spermidine concentrations declined with time in the tissues grown on maturation medium spermine was present in only minute quantities and showed only a small change with time. The presence of difluoromethylomithine in the culture medium had little effect on polyamine concentration, whereas the presence of difluoromethylarginine caused a decrease in putrescine concentrations in both red spruce and Norway spruce tissues grown on proliferation medium or maturation medium.

Abstract

Effects of various partial pressures of oxygen (5, 20 and 45 kPa) and carbon dioxide (0.03 and 6 kPa) on initiation, proliferation and maturation of somatic embryos in Picea abies were studied. The pO2 had a significant effect on the initiation of embryogenic tissue from mature zygotic embryos. However, the effect of pO2 was dependent on the strength of the basal medium.Low pO2 stimulated the formation of embryogenic tissue when the zygotic embryos were incubated on full strength medium, but was inhibitory when half-strength medium was used.Proliferation of embryogenic tissue was stimulated by higher partial pressures of both CO2 and O2. The effect of the gas phase on maturation of somatic embryos varied between different cell lines. However, there was a general tendency for 5 kPa O2 and 6 kPa CO2 to stimulate maturation.