Paal Krokene

Research Professor

(+47) 995 16 013
paal.krokene@nibio.no

Place
Ås H8

Visiting address
Høgskoleveien 8, 1433 Ås

Abstract

Bark beetles and their symbiotic bluestain fungi kill more trees than all other natural factors and cause great economic losses in Norway spruce and other conifers. The tree's natural defenses are the most important factor maintaining bark beetle-fungus complexes at low, endemic levels. Spraying Norway spruce trees with the plant hormone methyl jasmonate (MeJA) primes tree defenses without eliciting notable induced defenses, but enables the trees to respond much more quickly and strongly when challenged by bark beetles or fungi several weeks after treatment. This phenomenon, known as defense priming, is a form of acquired resistance that enables cost-effective and vigorous defense responses. In field experiments with 50-year-old clonal spruce trees terpene concentrations in the bark increased 60-fold within 24 h after mechanical wounding of MeJA primed trees, compared with a 13-fold increase in unprimed control trees. We also observed altered transcriptional patterns in primed trees using Illumina deep transcriptome sequencing. When wounded, primed trees launched vigorous induced defenses with significant differential regulation of gene transcripts, such as those involved in phenylpropanoid synthesis leading to lignification. Resistance-like genes, such as the NB-LRR coding genes, are also more rapidly induced in primed than in unprimed trees. Transcriptome results from primed but unwounded trees indicate an alteration in the state of the chromatin, resembling changes associated with the activity of the epigenetic machinery creating long-lasting epigenetic marks. We do not know yet how long the primed state is activated in Norway spruce, but our data so far indicate that it may last for at least 3 years.

Abstract

1 The European spruce bark beetle Ips typographus is a damaging pest on spruce in Europe. Beetle interactions with tree species originating outside the natural range of the beetle are largely unknown and may be unpredictable because trees without a co-evolutionary history with the beetle may lack effective defences. 2 The terpenoid composition and breeding suitability for I. typographus of the historic host Norway spruce Picea abies were compared with two evolutionary naïve spruces of North American origin that are extensively planted in North-West Europe: Sitka spruce Picea sitchensis and Lutz spruce Picea glauca x lutzii. 3 The bark of all three species had a similar chemical composition and similar levels of total constitutive terpenoids, although Norway spruce had higher total induced terpenoid levels. 4 Beetles tunnelling in the three spruce species produced similar amounts of aggregation pheromone. Controlled breeding experiments showed that I. typographus could produce offspring in all three species, with a similar offspring length and weight across species. However, total offspring production was much lower in Sitka and Lutz spruce. 5 Overall, the results of the present study suggest that I. typographus will be able to colonize Sitka and Lutz spruce in European plantations and in native spruce forests in North America if introduced there.

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Abstract

Global warming will most likely lead to increased drought stress in forest trees. We wanted to describe the adaptive responses of fine roots and fungal hyphae, at different soil depths, in a Norway spruce stand to long-term drought stress induced by precipitation exclusion over two growing seasons. We used soil cores, minirhizotrons and nylon meshes to estimate growth, biomass and distribution of fine roots and fungal hyphae at different soil depths. In control plots fine roots proliferated in upper soil layers, whereas in drought plots there was no fine root growth in upper soil layers and roots mostly occupied deeper soil layers. Fungal hyphae followed the same pattern as fine roots, with the highest biomass in deeper soil layers in drought plots. We conclude that both fine roots and fungal hyphae respond to long-term drought stress by growing into deeper soil layers.

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Abstract

The purpose of this study is to increase the basic understanding of outbreak dynamics in order to improve the management of bark beetle outbreaks. The spruce bark beetle Ips typographus is a major disturbance agent of European forests and is the continent’s most economically and environmentally damaging bark beetle. Outbreaks of the spruce bark beetle are often triggered by large windfall episodes, and we have utilized a unique opportunity to study a Slovakian outbreak where little salvage logging was performed in some areas after a 2.5 million m3 storm-felling in 2004. Our analyses focused on the first five years after the windfall, and we used a combination of empirical data and simulation models to understand the spatial patterns of beetle-killed forest patches developing during the outbreak. The univoltine beetle population used an increasing proportion of the windfelled trees during the two first seasons after the storm, but from the third season onwards our comparisons of inter-patch distance distributions indicated a transition from beetle production largely in windfall areas to a self-sustaining outbreak with infestation patches developing independently of the windthrows. The size of new infestation patches formed after this transition was modeled as a function of beetle pressure, estimated by the proportion of a circle area surrounding new patches that was covered by infestation patches the previous year. Our model results of patch size distribution did not correspond well with the empirical data if patch formation was modeled as a pure dispersal–diffusion process. However, beetle aggregation on individual trees appears to be important for patch development, since good correspondence with empirical data was found when beetle aggregation was incorporated in the modeled dispersal process. The strength of correspondence between the beetle aggregation model and the empirical data varied with the density of aggregation trees in the modeled landscape, and reached a maximum of 83% for a density of three aggregation trees per infestation patch. Our results suggest that efficient removal of windfelled trees up until the start of the second summer after a major windfall is important to avoid a transition into a patch-driven bark beetle outbreak that is very difficult to manage. Our results also indicate that the outcome of a patch-driven outbreak is difficult to predict, since the development of new infestation patches is not a simple function of beetle pressure but is also affected by beetle behavior and local forest conditions.

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Abstract

Trees must respond to many environmental factors during their development, and light is one of the main stimuli regulating tree growth. Thinning of forest stands by selective tree removal is a common tool in forest management that increases light intensity. However, morphological and anatomical adaptations of individual shoots to the new environmental conditions created by thinning are still poorly understood. In this study, we evaluated shoot morphology (shoot length, needle number, projected leaf area) and anatomy (tracheid lumen area, tracheid number, tracheid dimensions, xylem area, potential hydraulic conductivity) in three Norway spruce (Picea abies/L./Karst.) families exposed to different thinning regimes. We compared shoot characteristics of upper-canopy (i.e. sun-exposed) and lower-canopy (i.e. shaded) current-year shoots in a control plot and a plot thinned to 50 % stand density the previous year. One tree per family was chosen in each treatment, and five shoots were taken per canopy position. We found that upper-canopy shoots in both plots had higher values than lower-canopy shoots for all studied parameters, except lumen roundness and tracheid frequency (i.e. tracheid number per xylem area). Thinning had little effect on shoot morphology and anatomy 1 year after thinning, except for small but significant changes in tracheid dimensions. Needles were more sensitive to altered light conditions, as projected leaf area of shoot, needle number and leaf hydraulic conductivity changed after thinning. Differences between upper- and lower-canopy shoots did not seem to be influenced by thinning and were almost the same in both plots. Our results suggest that lower-canopy shoots require several years to modify their morphology and anatomy to new light conditions following thinning. The slow light adaptation of the lower canopy may be of practical importance in forest management: thinned stands may be predisposed to drought stress because newly exposed shoots experience increased illumination and transpiration after thinning.

Abstract

Increasing inter-continental trade with wood chips represents a challenge for phytosanitary authorities, as such trade may lead to pest introductions and invasions with huge impacts on forest ecosystems and economy. Predicting species invasions and their impacts in advance may be difficult, but improved information about potential invasive species ahead of any interceptions is an important precautionary step to reduce the probability of invasions. Here we identify bark- and wood-boring insects that have a potential to become invasive in northern Europe, and that may be introduced by import of deciduous wood chips from North America. The potentially most damaging species belong to the beetle genus Agrilus (Buprestidae), which includes the highly damaging emerald ash borer A. planipennis. We give a brief presentation of this and seven other Agrilus species or subspecies, and review factors of importance for the risk of establishment and potential economic and ecological impacts of these species. We also discuss one Scolytinae, Hylurgopinus rufipes. There are strong indications in the literature that some north European trees are highly susceptible to attack from the selected beetle species. We therefore conclude that because north European trees have not coevolved with these herbivores and thus may lack adequate defenses, most of the identified beetle species are likely to spread in “defense- and enemy-free space” if they are introduced to northern Europe, with considerable economic and ecological consequences.

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Abstract

Bark beetles and associated fungi are among the greatest natural threats to conifers worldwide. Conifers have potent defenses, but resistance to beetles and fungal pathogens may be reduced if tree stored resources are consumed by fungi rather than used for tree defense. Here, we assessed the relationship between tree stored resources and resistance to Ceratocystis polonica , a phytopathogenic fungus vectored by the spruce bark beetle Ips typographus. We measured phloem and sapwood nitrogen, non-structural carbohydrates (NSC), and lipids before and after trees were attacked by I. typographus (vectoring C. polonica) or artificially inoculated with C. polonica alone. Tree resistance was assessed by measuring phloem lesions and the proportion of necrotic phloem around the tree’s circumference following attack or inoculation. While initial resource concentrations were unrelated to tree resistance to C. polonica, over time, phloem NSC and sapwood lipids declined in the trees inoculated with C. polonica. Greater resource declines correlated with less resistant trees (trees with larger lesions or more necrotic phloem), suggesting that resource depletion may be caused by fungal consumption rather than tree resistance. Ips typographus may then benefit indirectly from reduced tree defenses caused by fungal resource uptake. Our research on tree stored resources represents a novel way of understanding bark beetle-fungal-conifer interactions.

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Abstract

Purpose: Drought-induced tree susceptibility is a major risk associated with climate change. Here we report how an 11-week drought affected tracheid structure, gene expression, and above- and belowground growth in 5-year-old Norway spruce trees (Picea abies) under controlled conditions. Results: The canopy of trees subjected to severe drought had significantly less current-year needle biomass, and fewer tracheids and tracheid rows in current-year shoots compared to fully watered control trees. Belowground tissues were more strongly affected by drought than aboveground tissues. In fine roots (<2 mm diameter) severe drought significantly reduced root biomass, root diameter, root length density and root surface area per soil volume compared to the control. Tracheid diameter and hydraulic conductivity in fine roots were significantly lower and tracheid flatness higher in trees subjected to severe drought than in control trees, both for long and short roots. Transcripts of the drought-related dehydrins PaDhn1 and PaDhn6 were strongly upregulated in stem bark and current-year needles in response to drought, whereas PaDhn4.5 was down-regulated. Conclusions: This study demonstrates that drought reduces biomass and hydraulic conductivity in fine roots and needles. We suggest that the ratio between PaDhn6 and PaDhn4.5 may be a sensitive marker of drought stress in Norway spruce.

Abstract

1. Neodiprion sertifer nucleopolyhedrovirus (NeseNPV) is widely used as a viral bio-insecticide against larvae of the European pine sawfly N. sertifer (Geoff.) (Hymenoptera: Diprionidae), which is one of the most harmful defoliators of pines in Northern Europe. A major obstacle to studying this pathogenic virus in nature is the difficulty of confirming and quantifying the presence of NeseNPV. 2. In the present study, we developed real-time polymerase chain reaction (PCR) primers, based on the caspid gene 39 sequence, for the specific and quantitative detection of NeseNPV. The quantitative real-time PCR (qPCR) assay can detect virus from any substrate tested, including different insect life stages (egg, larval, adult), pine foliage, and litter or ground vegetation. The reproducible detection limit for the real-time assay is 0.013 pg of viral DNA (0.013 × 10−12 g), corresponding to 136 viral genomes or approximately one to seven virus occlusion bodies per sample. 3. qPCR is a specific, quantitative, sensitive, reliable and flexible procedure, and is a good supplement to conventional microscopy- or bioassay-based methods for detection of the virus. We have used qPCR to quantify the level of NeseNPV in samples collected in the field after aerial application of the virus, and demonstrated significantly higher virus levels in sawfly larvae from sprayed areas compared with unsprayed control areas 4 weeks after spraying. 4. This qPCR assay can be used to determine important aspects of the biology of NeseNPV (e.g. virus levels in different insect life stages and in their microhabitats on pine foliage and in forest litter).

Abstract

Insects are among the organisms that will react most rapidly to climate change. They have a short generation span, they are very mobile, and their rate of development is directly impacted by temperature. This means that the development from egg to adult insect is much quicker when temperatures rise – a fact that may bode bad news for forestry.

Abstract

This chapter provides an overview of anatomical and ecological aspects of resin-based defences in pines and contrasts the defence strategy of pines with that of other conifers. The main constituents of conifer resin are mono- and diterpenes in about equal amounts, with smaller amounts of sesquiterpenes. Resin production and storage represent a great cost for the trees, and because resin is both chemically toxic and physically deterring to insects and pathogens it has long been considered an important defence mechanism in conifers. Preformed or constitutive resin structures are present in pines and all other members of the pine family, but are generally absent in non-Pinaceae species. Resin stored under pressure in constitutive ducts flows out when a tree is injured and helps trapping or repelling invading organisms and sealing the wound. Pines have constitutive resin ducts in needles, phloem and xylem. In the phloem and xylem constitutive resin ducts are oriented both radially (within the radial rays) and axially in the form of cortical resin ducts in the outer phloem and constitutive resin ducts in the xylem. Numerous connections between the radial resin ducts and the axial resin ducts in the xylem create a large inter-connected resin reservoir. In addition, so-called traumatic resin ducts can be induced axially in the xylem in response to wounding, insect attack or other biotic and abiotic stresses. Traumatic resin ducts may contribute to so-called acquired or systemic induced resistance that increases tree resistance to future attacks.

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

Ceratocystis polonica and Heterobasidion parviporum are important fungal pathogens in Norway spruce (Picea abies). Tree susceptibility to these pathogens with respect to phenology was studied using artificial fungal inoculations at six stages of bud development, and assessed by measuring phloem necroses in the stems of 2- and 8-year-old trees. Tree capacity for resistance was assessed by measuring phloem nonstructural carbohydrates at each stage. Phloem necroses were significantly larger in trees with fungal versus control inoculations and increased significantly over time. Changes in nonstructural carbohydrates occurred in the trees; a significant decline in starch and a slight but significant increase in total sugars occurred over time. These results suggest that susceptibility to fungal pathogens and carbohydrate levels in the stems of the trees were related to fine-scale changes in bud development. A trade-off may occur between allocation of starch (the major fraction of the stem carbohydrate pool) to bud development/shoot growth versus defence of the stem. Previous tests of plant defence hypotheses have focused on herbivory on plants growing under different environmental conditions, but the role of phenology and the effect of pathogens are also important to consider in understanding plant resource allocation patterns.

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Abstract

Conifer needles are extraordinarily variable and much of this diversity is linked to the water transport capacity of the xylem and to xylem conduit properties. However, we still know little about how anatomical characteristics influence the hydraulic efficiency of needle xylem in different parts of the crown. In this study we evaluated needle function and anatomy in Norway spruce families exposed to different light conditions. We measured tracheid and needle characteristics of sun-exposed and shaded current-year needles in two experimental plots: a control plot and a thinned plot with 50% reduction in stand density. Sun-exposed needles had a larger tracheid lumen area than shaded needles, and this was caused by a larger maximum tracheid lumen diameter, while the minimum lumen diameter was less plastic. Sun-exposed needles had also higher theoretical hydraulic conductivity than shaded needles. Thinning leads to increased radiation to the lower branches, and presumably exposes the upper branches to stronger water stress than before thinning. Thinning affected several needle parameters both in sun-exposed and shaded needles; tracheid lumens were more circular and minimum tracheid lumen diameter was larger in the thinned plot, whereas maximum tracheid lumen diameter was less plastic on both plots. This study demonstrates that needle xylem structure in Norway spruce is clearly influenced by the light gradient within the tree crown.

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Abstract

Secondary attraction to aggregation pheromones plays a central role in the host colonization behavior of the European spruce bark beetle Ips typographus. However, it is largely unknown how the beetles pioneering an attack locate suitable host trees, and eventually accept or reject them. To find possible biomarkers for host choice by I. typographus, we analyzed the chemistry of 58 Norway spruce (Picea abies) trees that were subsequently either (1) successfully attacked and killed, (2) unsuccessfully attacked, or (3) left unattacked. The trees were sampled before the main beetle flight in a natural Norway spruce-dominated forest. No pheromones were used to attract beetles to the experimental trees. To test the trees’ defense potential, each tree was treated in a local area with the defense hormone methyl jasmonate (MeJ), and treated and untreated bark were analyzed for 66 different compounds, including terpenes, phenolics and alkaloids. The chemistry of MeJ-treated bark correlated strongly with the success of I. typographus attack, revealing major chemical differences between killed trees and unsuccessfully attacked trees. Surviving trees produced significantly higher amounts of most of the 39 analyzed mono-, sesqui-, and diterpenes and of 4 of 20 phenolics. Alkaloids showed no clear pattern. Differences in untreated bark were less pronounced, where only 1,8-cineole and (−)-limonene were significantly higher in unsuccessfully attacked trees. Our results show that the potential of individual P. abies trees for inducing defense compounds upon I. typographus attack may partly determine tree resistance to this bark beetle by inhibiting its mass attack.

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.

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Abstract

Norway spruce (Picea abies) bark contains specialized phloem parenchyma cells that swell and change their contents upon attack by the bark beetle Ips typographus and its microbial associate, the blue stain fungus Ceratocystis polonica. These cells exhibit bright autofluorescence after treatment with standard aldehyde fixatives, and so have been postulated to contain phenolic compounds. Laser microdissection of spruce bark sections combined with cryogenic NMR spectroscopy demonstrated significantly higher concentrations of the stilbene glucoside astringin in phloem parenchyma cells than in adjacent sieve cells. After infection by C. polonica, the flavonoid (+)-catechin also appeared in phloem parenchyma cells and there was a decrease in astringin content compared to cells from uninfected trees. Analysis of whole-bark extracts confirmed the results obtained from the cell extracts and revealed a significant increase in dimeric stilbene glucosides, both astringin and isorhapontin derivatives (piceasides A to H), in fungus-infected versus uninfected bark that might explain the reduction in stilbene monomers. Phloem parenchyma cells thus appear to be a principal site of phenolic accumulation in spruce bark.

Abstract

Damage by cone and seed insects is a severe impediment to those producing conifer seeds in seed orchards. The SNS-supported network `Nordic cone and seed insects` has been cooperating to address this problem since 2007. One of the outcomes is the coordinated monitoring of the most important insect species. The data collected form a basis for establishing strategies for pest management.

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Abstract

Conifers and other trees are constantly adapting to changes in light conditions, water/nutrient supply and temperatures by physiological and morphological modifications of their foliage. However, the relationship between physiological processes and anatomical characteristics of foliage has been little explored in trees. In this study we evaluated needle structure and function in Norway spruce families exposed to different light conditions and transpiration regimes. We compared needle characteristics of sun-exposed and shaded current-year needles in a control plot and a thinned plot with 50% reduction in stand density. Whole-tree transpiration rates remained similar across plots, but increased transpiration of lower branches after thinning implies that sun-exposed needles in the thinned plot were subjected to higher water stress than sun-exposed needles in the control plot. In general, morphological and anatomical needle parameters increased with increasing tree height and light intensity. Needle width, needle cross-section area, needle stele area and needle flatness (the ratio of needle thickness to needle width) differed most between the upper and lower canopy. The parameters that were most sensitive to the altered needle water status of the upper canopy after thinning were needle thickness, needle flatness and percentage of stele area in needle area. These results show that studies comparing needle structure or function between tree species should consider not only tree height and light gradients, but also needle water status. Unaccounted for differences in needle water status may have contributed to the variable relationship between needle structure and irradiance that has been observed among conifers.

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Abstract

We treated Norway spruce (Picea abies) stems with methyl jasmonate (MeJA) to determine possible quantitative and qualitative eVects of induced tree defenses on pheromone emission by the spruce bark beetle Ips typographus. We measured the amounts of 2-methyl-3-buten-2-ol and (S)-cis-verbenol, the two main components of the beetle’s aggregation pheromone, released from beetle entrance holes, along with phloem terpene content and beetle performance in MeJA-treated and untreated Norway spruce logs. As expected, phloem terpene levels were higher and beetle tunnel length was shorter (an indication of poor performance) in MeJA-treated logs relative to untreated logs. Parallel to the higher phloem terpene content and poorer beetle performance, beetles in MeJA-treated logs released signiWcantly less 2-methyl-3-buten-2-ol and (S)-cis-verbenol, and the ratio between the two pheromone components was signiWcantly altered. These results suggest that host resistance elicited by MeJA application reduces pheromone emission by I. typographus and alters the critical ratio between the two main pheromone components needed to elicit aggregation. The results also provide a mechanistic explanation for the reduced performance and attractivity observed in earlier studies when bark beetles colonize trees with elicited host defenses, and extend our understanding of the ecological functions of conifer resistance against bark beetles.

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Abstract

Background: Tree-killing bark beetles (Coleoptera, Scolytinae) are among the most economically and ecologically important forest pests in the northern hemisphere. Induction of terpenoid-based oleoresin has long been considered important in conifer defense against bark beetles, but it has been difficult to demonstrate a direct correlation between terpene levels and resistance to bark beetle colonization. Methods: To test for inhibitory effects of induced terpenes on colonization by the spruce bark beetle Ips typographus (L.) we inoculated 20 mature Norway spruce Picea abies (L.) Karsten trees with a virulent fungus associated with the beetle, Ceratocystis polonica (Siem.) C. Moreau, and investigated induced terpene levels and beetle colonization in the bark. Results: Fungal inoculation induced very strong and highly variable terpene accumulation 35 days after inoculation. Trees with high induced terpene levels (n = 7) had only 4.9% as many beetle attacks (5.1 vs. 103.5 attacks m22) and 2.6% as much gallery length (0.029 m m22 vs. 1.11 m m22) as trees with low terpene levels (n = 6). There was a highly significant rank correlation between terpene levels at day 35 and beetle colonization in individual trees. The relationship between induced terpene levels and beetle colonization was not linear but thresholded: above a low threshold concentration of ,100 mg terpene g21 dry phloem trees suffered only moderate beetle colonization, and above a high threshold of ,200 mg terpene g21 dry phloem trees were virtually unattacked. Conclusion/Significance: This is the first study demonstrating a dose-dependent relationship between induced terpenes and tree resistance to bark beetle colonization under field conditions, indicating that terpene induction may be instrumental in tree resistance. This knowledge could be useful for developing management strategies that decrease the impact of tree-killing bark beetles.

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Abstract

Norway spruce (Picea abies) defends itself against herbivores and pathogens by formation of traumatic resin ducts filled with terpenoid-based oleoresin. An important group of enzymes in terpenoid biosynthesis are the short-chain isoprenyl diphosphate synthases which produce geranyl diphosphate (C10), farnesyl diphosphate (C15), and geranylgeranyl diphosphate (C20) as precursors of monoterpenes, sesquiterpenes, and diterpene resin acids, respectively. After treatment with methyl jasmonate (MJ) we investigated the expression of all isoprenyl diphosphate synthase genes characterized to date from Norway spruce and correlated this with formation of traumatic resin ducts and terpene accumulation. Formation of traumatic resin ducts correlated with higher amounts of monoterpenes, sesquiterpenes and diterpene resin acids and an upregulation of isoprenyl diphosphate synthase genes producing geranyl diphosphate or geranylgeranyl diphosphate. Among defense hormones, jasmonate and jasmonate-isoleucine conjugate accumulated to higher levels in trees with extensive traumatic resin duct formation, whereas salicylate did not. Jasmonate and ethylene are likely to both be involved in formation of traumatic resin ducts based on elevated transcripts of genes encoding lipoxygenase and 1-aminocyclopropane-1-carboxylic acid oxidase associated with resin duct formation. Other genes involved in defense signalling in other systems, mitogen-activated protein kinase3 and nonexpressor of pathogenesis-related gene1, were also associated with traumatic resin duct formation. These responses were detected not only at the site of MJ treatment, but also systemically up to 60 cm above the site of treatment on the trunk.

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Abstract

The Eurasian spruce bark beetle, Ips typographus, is one of the major forest insect pests in Europe, capable of mass-attacking and killing mature Norway spruce trees. The initiation and development of a new generation are strongly controlled by temperature and a warmer climate may affect the number of generations that is produced per year and hence the outbreak dynamics. Experimental knowledge regarding reproductive diapause adaptations is, however, too sparse for largescale assessments of future trends. We developed a model description of diapause induction, and used gridded observational temperature data to evaluate multiple combinations of day length and temperature thresholds to find the model parameterisation most coherent with I. typographus monitoring data from Scandinavia. The selected model parameterisation is supported by European literature data, though further experimental studies are required to analyse population specific adaptations and capacity for adjustments to changing climate conditions. Implementing the model description of reproductive diapause in a temperature driven model of bark beetle phenology (swarming activity and development from egg to mature bark beetle), enabled us to assess the length of the late summer swarming period that is a critical determinant of the risk of forest damage. By using regional climate model data we show that higher temperatures can result in increased frequency and length of late summer swarming events, producing a second generation in southern Scandinavia and a third generation in lowland parts of central Europe. Reproductive diapause will not prevent the occurrence of an additional generation per year, but the day length cues may restrict the length of the late summer swarming period.

Abstract

Today the spruce bark beetle Ips typographus is always univoltine in Northern Europe including Norway and completes development from egg to adult between May and August. Further south in Europe, development is bivoltine with the completion of two generations in most years. A temperature-driven developmental model suggests that by 2070-2100 the voltinism of I. typographus will change dramatically in Norway. If summers become only 2.5°C warmer than today bivoltinism can be expected every single year in the major spruce growing areas in S-Norway. This is likely to have dramatic effects on forestry since two generations per year will give two, instead of one, attack periods each summer. In addition to increasing the number of attacked trees the effect of the attacks may also be more severe, as Norway spruce is more susceptible to beetle attacks later in the summer. However, climate change will probably also change the phenology of Norway spruce and thus its susceptibility to attack by I. typographus and its phytopathogenic fungal associates. We are currently modelling how tree resistance varies with temperature and tree phenology in order to provide more well-founded advice to forest managers on the interaction between bark beetles and tree in a future climate.

Abstract

We monitored the effects of the drought stress on 20-year old clones of Norway spruce (Picea abies) by using a range of instrumental methods. On two experimental plots (Hoxmark, Norway, 59°40\"14`N, 10°47\"36`E) the drought was induced in a period between May and October 2009 by removing the throughfall using the rain shelters and trenching. We collected data on soil moisture, stem and branch sap flow, xylem diameter, anatomical and calorimetric analysis of the needles, fine root biomass and dynamics and resistance to pathogens. Standard meteorological data were collected locally throughout the whole period. Here we present the preliminary analysis of sap flow and xylem diameter in a period 1-17 august 2009. The sap flow was measured on stems in the breast height by using the method of stem tissue heat balance (THB, EMS Brno). The values were measured once in 2 minutes and saved as the average of 10 minutes. The fluctuations in xylem diameter was monitored by using the automatic dendrometers DR26 (EMS Brno). We evaluated both diurnal and seasonal dynamics. Preliminary results show a significant difference in shape of diurnal curves of transpiration as well as different time lag among the sap flow and the potential evapo-transpiration. Also the differences in diurnal dynamics of the stem circumference suggested different xylem water potential in stressed and control trees. In the drought-stressed trees the diurnal fluctuation in stem diameter was about 4 times higher and the total stem increment one third lower, com-pared to the control trees.

Abstract

Cone and seed insects reduce seed production in seed orchards by feeding on cones and/or seeds and thus cause a lack of seedlings for reforestation. One of the most serious pest species in conifers is the spruce cone worm, Dioryctria abietella. Since 2007, we have used pheromone traps to monitor the flight of this species in Denmark, Finland, Norway and Sweden (and Estonia from 2009). In addition to monitoring, all countries have measured air temperatures in the vicinity of the traps. It is thus possible to correlate flight activity with temperature and temperature sums (accumulated day degrees >5°C). In Sweden cone development has also been registered throughout the monitoring period. Our results show that D. abietella has an extended flight period in Northern Europe, which lasts from late May to late September. Further research is needed to determine if late flying individuals oviposit on shoots or in cones. Increased knowledge about the flight period of D. abietella and how it coincides with temperature and cone development may help us develop better control measures against this important pest.

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Abstract

The blue-stain fungus Ceratocystis resinifera colonizes wounds on living Picea spp. and other conifers in Europe and North America. Little is known regarding the pathogenicity of this fungus and consequently, four Norwegian C. resinifera isolates were inoculated on to Norway spruce (Picea abies) using two different techniques. These included single-point inoculations on young trees (two inoculations per tree on 14-year-old trees) and mass-inoculations on older trees (∼200 inoculations per tree on 34-year-old trees). In both experiments, C. resinifera induced minor symptoms that in most cases did not differ significantly from inoculation with sterile agar. The virulent blue-stain fungus C. polonica, which was inoculated for comparative purposes, induced extensive symptoms, causing 83% dead cambium circumference and 82% blue-stained sapwood, and long necrotic lesions in the phloem. The results suggest that C. resinifera is non-pathogenic or only mildly pathogenic to Norway spruce and does not present a threat to these trees.

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Abstract

Constitutive and inducible terpene production is involved in conifer resistance against bark beetles and their associated fungi. In this study 72 Norway spruce (Picea abies) were randomly assigned to methyl jasmonate (MJ) application, inoculation with the bluestain fungus Ceratocystis polonica, or no-treatment control. We investigated terpene levels in the stem bark of the trees before treatment, 30 days and one year after treatment using GC–MS and two-dimensional GC (2D-GC) with a chiral column, and monitored landing and attack rates of the spruce bark beetle, Ips typographus, on the trees by sticky traps and visual inspection. Thirty days after fungal inoculation the absolute amount and relative proportion of (+)-3-carene, sabinene, and terpinolene increased and (+)-α-pinene decreased. Spraying the stems with MJ tended to generally increase the concentration of most major terpenes with minor alteration to their relative proportions, but significant increases were only observed for (−)-β-pinene and (−)-limonene. Fungal inoculation significantly increased the enantiomeric ratio of (−)-α-pinene and (−)-limonene 1 month after treatment, whereas MJ only increased that of (−)-limonene. One year after treatment, both MJ and fungal inoculation increased the concentration of most terpenes relative to undisturbed control trees, with significant changes in (−)-β-pinene, (−)-β-phellandrene and some other compounds. Terpene levels did not change in untreated stem sections after treatment, and chemical induction by MJ and C. polonica thus seemed to be restricted to the treated stem section. The enantiomeric ratio of (−)-α-pinene was significantly higher and the relative proportions of (−)-limonene were significantly lower in trees that were attractive to bark beetles compared to unattractive trees. One month after fungal inoculation, the total amount of diterpenes was significantly higher in putative resistant trees with shorter lesion lengths than in putative susceptible trees with longer lesions. Thus, terpene composition in the stem bark may be related to resistance of Norway spruce against I. typographus and C. polonica.

Abstract

Constitutive and inducible terpene production is involved in conifer resistance against bark beetles and their associated fungi. In this study 72 Norway spruce (Picea abies) were randomly assigned to methyl jasmonate (MJ) application, inoculation with the bluestain fungus Ceratocystis polonica, or no-treatment control. We investigated terpene levels in the stem bark of the trees before treatment, 30 days and one year after treatment using GC–MS and two-dimensional GC (2D-GC) with a chiral column, and monitored landing and attack rates of the spruce bark beetle, Ips typographus, on the trees by sticky traps and visual inspection. Thirty days after fungal inoculation the absolute amount and relative proportion of (+)-3-carene, sabinene, and terpinolene increased and (+)-α-pinene decreased. Spraying the stems with MJ tended to generally increase the concentration of most major terpenes with minor alteration to their relative proportions, but significant increases were only observed for (-)-β-pinene and (-)-limonene. Fungal inoculation significantly increased the enantiomeric ratio of (-)-α-pinene and (-)-limonene 1 month after treatment, whereas MJ only increased that of (-)-limonene. One year after treatment, both MJ and fungal inoculation increased the concentration of most terpenes relative to undisturbed control trees, with significant changes in (-)-β-pinene, (-)-β-phellandrene and some other compounds. Terpene levels did not change in untreated stem sections after treatment, and chemical induction by MJ and C. polonica thus seemed to be restricted to the treated stem section. The enantiomeric ratio of (-)-α-pinene was significantly higher and the relative proportions of (-)-limonene were significantly lower in trees that were attractive to bark beetles compared to unattractive trees. One month after fungal inoculation, the total amount of diterpenes was significantly higher in putative resistant trees with shorter lesion lengths than in putative susceptible trees with longer lesions. Thus, terpene composition in the stem bark may be related to resistance of Norway spruce against I. typographus and C. polonica.

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Abstract

The anamorph genus Leptographium Lagerberg and Melin includes species that are typically bark beetleassociated fungi, with teleomorphs in Grosmannia. During a survey of ophiostomatoid fungi in Norway, two unusual species, that fit the broader morphological description of Leptographium, were isolated directly from the rootfeeding beetles, Dryocetes authographus and Hylastes cunicularius, as well as from roots infested by these insects. The first of these could be distinguished from other described species based on a sparse sporulation, black spore drops and chlamydospores in older cultures. This species also produces a Hyalorhinocladiella synanamorph. The second species was characterised by distinctly curved conidia. Based on these unusual morphological characteristics and distinct DNA sequences, these fungi were recognised as new taxa for which the names Leptographium chlamydatum sp. nov. and L. curvisporum sp. nov. are provided.

Abstract

The interaction between conifers, ophiostomatoid fungi and bark beetles is a key factor in conifer ecosystems worldwide, since combined beetle-fungus attacks may cause massive tree mortality, huge economical losses and landscape-level ecological changes. For more than a century researchers have been arguing about who is responsible for tree mortality in bark beetle attacked trees - the beetles themselves or their associated fungi. As in many such polarized debates the best answer probably lies somewhere in between the extremes. The beetles are obviously central in tree killing, as they are actively selecting suitable host trees, short-circuiting tree defenses by boring straight into the relatively defenseless cambial area, and causing mechanical damage to the phloem. However, ophiostomatoid fungi are equally obvious contributors to tree death since nearly all tree-killing bark beetles are associated with such fungi, these fungi are able to colonize and kill healthy phloem and sapwood far beyond the beetle tunnels, and many fungi can kill healthy trees in experimental mass-inoculations. Thus, the trees are facing a beetle-fungus complex that probably acts synergistically to overwhelm tree defenses. The beetles’ mass-attack strategy is central to the success of the beetle-fungus complex, since multiple attacks deplete tree defenses and speed up tree-killing.

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

Combined bark beetle-fungus attacks are a key factor in conifer ecosystems in the Northern Hemisphere, where they regularly cause massive tree mortality. Central to the success of the bark beetle-fungus complex is the fact that bark beetles are among the few insects that are capable of breaching the potent anatomical and chemical defenses of healthy tree stems.....

Abstract

Tree-killing bark beetles conquer healthy trees by means of pheromone mediated mass-attacks. The initial attackers select trees through a process of random landing and processing of tactile and short-range chemical stimuli. Inside acceptable hosts they produce aggregation pheromones that attract additional attackers. In a recent field experiment we induced defense responses in 60-year-old Norway spruce trees and monitored colonization by the spruce bark beetle Ips typographus. Induced stem sections had significantly less beetle colonization than control sections, with shorter parental galleries and fewer eggs deposited. In addition, fewer beetles were attracted to beetles tunnelling in induced bark. This reduced ability to attract conspecifics suggests that induced host defenses interfere with beetle pheromone production or emission. The mechanisms responsible for this are unknown, but may involve emission of volatile terpenes such as (E)-b-farnesene and linalool, which have been shown to increase up to 100-fold in induced Norway spruce trees. These compounds have been reported to attract natural enemies of herbivores or repel herbivores directly in other conifer-insect interactions.

Abstract

Seed production in Norway spruce (Picea abies) seed orchards may be severely reduced by insects attacking cones and seeds. Insects in several orders and genera are responsible for the damage, and at present there are no effective and environmentally acceptable control options. Studies in Sweden have shown promising results with spraying Bt (Bacillus thuringiensis var. kurstaki x aizawai) against species of Lepidoptera, but for other pests other control methods must be developed....

Abstract

The European pine sawfly Neodiprion sertifer is a widely distributed defoliator of pines that can cause substantial growth losses over extensive areas. It attacks most species of twoneedle pines in its distribution area, and have occasional short-lived outbreaks that may cover up to 200.000 ha. In Norway we have had outbreak populations in the eastern part of the country since 2004, and in an ongoing research project we are evaluating aerial application of the Neodiprion sertifer nuclear polyhedrosis virus (NsNPV) to control Neodiprion sertifer....

Abstract

Host tree terpenes can influence attraction of conifer-infesting bark beetles to their aggregation pheromones, and both synergistic and inhibitory effects have been reported. We tested a gradient of ratios of (–)-α-pinene, the predominant monoterpene in Norway spruce, to the pheromone of Ips typographus, a major pest of Norway spruce. Attraction of I. typographus increased as the release rate of (–)-α-pinene increased. The two highest (–)-α-pinene : pheromone ratios (526 : 1 and 2595 : 1) attracted twice as many I. typographus as pheromone alone, whereas low to intermediate ratios (56 : 1, 274 : 1) did not differ from pheromone alone. Our results are in agreement with a proposed model, which suggests that bark beetles display unique response profiles to host terpenes depending on the physiological condition of the host trees that they typically colonize. Ips typographus, which is an aggressive species capable of colonizing and killing healthy trees, showed an increased attraction to monoterpene : pheromone ratios, and this may be high enough to inhibit attraction of less aggressive beetle species typically colonizing dead, dying or stressed trees. Attraction of associates of I. typographus was also modified by (–)-α-pinene. Ips duplicatus, a competitor of I. typographus, showed increased attraction to the pheromone of I. typographus across all concentrations of (–)-α-pinene.

Abstract

The Norwegian Monitoring Programme for Forest Damage (OPS) has since its start registered damage to selected trees. The aim of the registrations has been to explain variations in crown density and crown colour. In answer to international requests, the Norwegian Forest and Landscape Institute has prepared a short guide to the determination of the most common forms of damage found in Norwegian forests...

Abstract

Model simulations show that an increased frequency in storms and drought periods may result in more frequent and shorter outbreaks of bark beetles. Warmer summers can result in two bark beetle generations per summer instead of one, giving bark beetles the opportunity to attack forests twice in a single year.

Abstract

Studies of defense mechanisms in Norway spruce Picea abies against the spruce bark beetle Ips typographus show that inducible defenses are triggered by unsuccessful beetle attacks. Moreover, spruce trees can be vaccinated against bark beetle attacks through stem bark inoculation with Ceratocystis polonica, an aggressive fungal associate of Ips typographus. Artificial inoculation with certain other fungi has a similar effect, making treated trees a tougher mach for attacking beetles. A similar enhancement of resistance can be achieved by stem surface treatment with methyl jasmonate. Enhanced resistance is associated with the formation of traumatic resin ducts, and mobilization of phenolic substances in living parenchyma cells. The phenolics appear to mix with the traumatic resin. Similar mechanisms are operating in other conifers. We hypothesize that unsuccessful beetle attacks produce a vaccination effect in the trees, enhancing their resistance to later attack. Adding the effect of induced resistance as a model factor shows an immediate effect on the population dynamics of Ips typographus.

Abstract

Bark beetles and associated phytopathogenic fungi elicit defence responses in conifers that may interfere with beetle establishment and development. Norway spruce is serving as a useful model species for studies of induced defences elicited by beetle attacks, fungal inoculation, and treatment with chemical elicitors.When trees are pretreated with a sublethal dose of fungal inoculations or with the phytohormone methyl jasmonate they become much more resistant to subsequent bark beetle attacks or artificial mass inoculations with fungi. This induced disease resistance follows dose-response dynamics, is nonspecific with respect to the pretreatment organism, appears to be nonsystemic, takes weeks rather than days to become activated, and can also be activated by mechanical wounding alone.Application of methyl jasmonate to Norway spruce stems induces a massive increase in terpene levels and external resin flow on the stem, whereas no increase is observed in soluble phenolics. Methyl jasmonate-application also leads to significantly less bark beetle colonization, with shorter parental galleries and fewer eggs laid in treated bark. There were also reductions in the number of beetles produced and the mean dry weight per beetle in methyl jasmonate-treated bark. Furthermore, fewer beetles were attracted to conspecifics tunneling in MJ-treated bark.The exact mechanisms responsible for induced resistance in Norway spruce and other conifers have not been determined, but inducible anatomical defense responses such as changes in polyphenol-containing parenchyma cells (PP cells) in the phloem and induction of traumatic resin duct formation in the sapwood seem to play an important role.

Abstract

When conifers such as Picea abies Karst. (Norway spruce) are attacked by insects or pathogens, they often respond by producing increased quantities of terpenoid oleoresin. This response can be mimicked in young P. abies seedlings by treatment with methyl jasmonate (MJ). In this study, we determined the effects of MJ on terpenoids and other chemical defenses of mature P. abies, and investigated whether this treatment protected trees against attack by the blue-stain fungus Ceratocystis polonica (Siem.) C. moreau, the most important fungal associate of the spruce bark beetle Ips typographus L. Methyl jasmonate treatment induced the formation of traumatic resin ducts in the developing xylem, enhanced resin flow and stimulated increased accumulation of monoterpenes, sesquiterpenes and diterpene resin acids. However, only minor changes were detected in terpene composition in response to MJ treatment and no changes in soluble phenolic concentration were measured. There was much variability in the timing and degree of response to MJ among clones. The observed chemical and anatomical changes in response to MJ treatment were correlated with increased resistance to C. polonica, suggesting that terpenoid oleoresin may function in defense against this pathogen.

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Abstract

When conifers such as Picea abies Karst. (Norway spruce) are attacked by insects or pathogens, they often respond by producing increased quantities of terpenoid oleoresin. This response can be mimicked in young P. abies seedlings by treatment with methyl jasmonate (MJ). In this study, we determined the effects of MJ on terpenoids and other chemical defenses of mature P. abies, and investigated whether this treatment protected trees against attack by the blue-stain fungus Ceratocystis polonica (Siem.) C. Moreau, the most important fungal associate of the spruce bark beetle Ips typo-graphus L. Methyljasmonate treatment induced the formation of traumatic resin ducts in the developing xylem, enhanced resin flow and stimulated increased accumulation of monoterpenes, sesquiterpenes and diterpene resin acids. However, only minor changes were detected in terpene composition in response to MJ treatment and no changes in soluble phenolic concentration were measured. There was much variability in the timing and degree of response to MJ among clones. The observed chemical and anatomical changes in response to MJ treatment were correlated with increased resistance to C. polonica, suggesting that terpenoid oleoresin may function in defense against this pathogen.

Abstract

The spruce bark beetle is the most serious pest of mature spruce trees in Eurasia. At low population densities it breeds in weakened or newly dead trees, but at high densities it becomes a tree killer. The exact factors triggering outbreaks are not fully understood, but climatic variables are important candidates. Populations in SE Norway have been monitored since 1979. Various time series methods are used to explore the role of climate in outbreaks of the spruce bark beetle in space and time, and to estimate the distribution of bivoltism under different scenarios of climate change. Large windfall events appear to be a major synchronizer of beetle outbreaks in areas subjected to regionalized weather systems, and the northern border of bivoltism may be moved towards north as a function of increasing temperature. Preliminary models of the population dynamics emphasize the frequency of large windfall events and phenological changes due to temperature increase. Final aims are to estimate the regional risks of forest damage under different scenarios of climate change, and to describe practices that may reduce the impact for forest managers.

Abstract

At least three mechanisms are known to cause synchrony among spatially separated insect populations: 1) dispersal among populations, 2) synchronous stochastic effects, often referred to as the Moran effect, and 3) trophic interactions with other species that are either themselves synchronized or mobile.The present study brings in the role of insect taxa for spatial synchrony. The spatial synchrony observed in several North American and Eurasian epidemic bark beetles was compared with patterns of synchrony in outbreaks of defoliating forest Lepidoptera, revealing a marked difference between these two major insect taxa.The bark beetles exhibited a generally lower degree of spatial synchrony than the Lepidoptera, possibly because bark beetles are synchronized by different weather variables that are acting on a smaller scale than those affecting the Lepidoptera, or because inherent differences in their dynamics leads to more cyclic oscillations and hence more synchronous spatial dynamics in the Lepidoptera. Among the epidemic bark beetles tested, spatial synchrony of outbreaks in the Eurasian spruce bark beetle Ips typographus was significantly higher than for the other species.

Abstract

The anatomical defense responses in stems of Norway spruce (Picea abies) clones of different resistance to pathogenic fungi were characterized over time and distance from small mechanical wounds or wounds inoculated with the root rot fungus Heterobasidion annosum. Common responses for both treatments included division of ray parenchyma and other cells in the cambial zone, accumulation of phenolic inclusions in ray parenchyma cells, activation of phloem parenchyma (PP) cells, and formation of traumatic resin ducts (TDs) in the xylem. TD formation occurred synchronously from a tangential layer of cells, or symplasmic domain, within the zone of xylem mother cells. TD induction is triggered by a signal, which propagates a developmental wave in the axial direction at about 2.5cm per day. TDs are formed at least 30cm above single inoculations within 16–36days after inoculation. The size and number of TDs is attenuated further away from the inoculation site, indicating a dose-dependent activity leading to TD development. Compared to sterile wounding, fungal inoculation gave rise to more and larger TDs in all clones, and multiple rows of TDs in weak clones. Fungal inoculation also induced the formation of more new PP cells, increasing the number of PP cells in the phloem in the year of inoculation up to 100%. TD and PP cell formation was greater in susceptible compared to resistant clones and after fungal versus sterile inoculation. Potential mechanisms responsible for this variable response are discussed.

Abstract

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.

Abstract

When conifers such as Picea abies (Norway spruce) are attacked by insects or pathogens, they often produce increased quantities of terpenoid oleoresin. This response can be mimicked in young P. abies seedlings by treatment with methyl jasmonate. In this study, we determined the effect of methyl jasmonate on the terpenoids and other chemical defenses of mature P. abies, and investigated if this treatment protected trees against attack by the blue-stain fungus Ceratocystis polonica, the most important fungal associate of the bark beetle Ips typographus. Methyl jasmonate treatment induced the formation of traumatic resin ducts in the developing xylem, enhanced resin flow, and stimulated increased accumulation of monoterpenes, sesquiterpenes, and diterpene resin acids. However, almost no significant changes in terpene composition were detected. In addition, no changes in soluble phenolic content were observed. There was a very high variability both among and within clones in the timing and degree of response to methyl jasmonate. These chemical and anatomical changes were correlated with increased resistance to C. polonica, suggesting that terpenoid oleoresin may function in defense against this pathogen.

Abstract

In 1996, 7000 ha of pine forests were defoliated by the pine looper Bupalus piniaria in south-western Sweden.The susceptibility of trees of different defoliation classes (0, 30, 60, 90 and 100% defoliation) to beetle-vectored blue-stain fungi was tested in inoculation experiments.Forty and 120-year-old Scots pine trees were inoculated with `single\", i.e. a few inoculations of Leptographium wingfieldii and Ophiostoma minus, two blue-stain fungi associated with the pine shoot beetle Tomicus piniperda. The young trees were also \"mass\" inoculated with L. wingfieldii at a density of 400 inoculation points per m2 over a 60 cm stem belt.Host tree symptoms indicated that only trees with 90100% defoliation were susceptible to the mass inoculation.Single inoculations did not result in any consistent differences in fungal performance between trees of different defoliation classes, regardless of inoculated species or tree age class.Leptographium wingfieldii produced larger reaction zones than O. minus, and both species produced larger lesions in old than in young trees.As beetle-induced tree mortality in the study area occurred only in totally defoliated stands, mass inoculations seem to mimic beetle-attacks fairly well, and thus seem to be a useful tool for assessing host resistance.As even severely defoliated pine trees were quite resistant, host defence reactions in Scots pine seem to be less dependent on carbon allocation than predicted by carbon-based defence hypotheses.

Abstract

The anatomical response of Norway spruce bark polyphenolic parenchyma cells (PP cells) to inoculation with the phytopathogenic fungus Ceratocystis polonica and attack by its bark-beetle vector Ips typographus was examined.Fungal inoculation on the periderm surface had no effect, while inoculation just below the periderm or halfway into the phloem (mid-phloem) generated detectable responses within 3 wk. The responses included increase in PP cell size and in periodic acid - Schiffs staining of PP cell phenolics, wound periderm initiation from PP cells, and cambial zone traumatic resin duct formation. Fungi were not seen in samples 3 wk after subperiderm or mid-phloem inoculation, but were found in some samples 6 and 9 wk after mid-phloem inoculation.In contrast, inoculations into the cambium resulted in partial (3 wk) or complete (6 and 9 wk) fungal colonization and death of tissue in the infected area. This indicates that PP cells have defenses capable of inhibiting fungal growth. Samples taken near bark-beetle galleries had similar anatomical responses as inoculated samples, validating the inoculation approach to studying defense responses in spruce.These results show that PP cells represent not only a constitutive defense system, but are also involved in local and remote inducible defenses against fungal and beetle attack.

Abstract

A field experiment was carried out to test the hypothesis that treatment of Norway spruce trees with the Ips typographus-transmitted blue-stain fungus Ceratocystis polonica enhances tree resistance to later mass attack by this bark beetle. Twenty-five mature trees were pre-treated by inoculating a non-lethal dose of the fungus into the bark, while 18 trees served as untreated controls. Three and a half weeks after treatment a bark beetle attack was initiated by attaching dispensers with I. typographus pheromone to the tree trunks. A significantly larger proportion (67%) of the control trees than of the pre-treated trees (36%) were killed by the beetle attack. The result is discussed in relation to recent results regarding defence mechanisms in Norway spruce trees.

Abstract

Norway spruce trees, exposed to sub-lethal attacks by the bark beetle Ips typographus, form distinct necrotic and resin-soaked reaction zones in the bark and outer sapwood within which the beetles and their associated fungi are enveloped and arrested. In these trees a ring of axial resin ducts regularly form in the stem xylem. These ducts are included in a false annual ring with narrow tracheids. A similar ring of resin ducts is seen in trees artificially administered a sub-lethal inoculation dose of the blue-stain fungus Ceratocystis polonica, a pathogenic associate of I. typographus. Here we report preliminary studies on the development of traumatic axial ducts in stems of Norway spruce after beetle and fungus invasion, and some cytochemical aspects related to the formation of phenolics and terpenes. We suggest that traumatic resin ducts furnish the reaction zones with materials toxic to both beetles and fungi, and that increased numbers of ducts may render trees more resistant to attack.

Abstract

Twenty-five year old Norway spruce trees (Picea abies) were inoculated with four blue-stain fungi. Each tree was inoculated three times with each fungus and three times with sterile agar as a control, giving a total of 15 inoculations per tree. There was little variation in the extent of phloem necrosis produced in response to the different fungi, but five weeks after inoculation necrosis induced by Ceratocystis polonica and Ambrosiella sp. were significantly longer than those for the other fungi. At the same time, C. polonica had induced sapwood desicc-ation twice as deeply into the wood as any other fungus.Hyphal growth of the fungi into phloem and sapwood followed the same pattern as necrosis length and desiccation depth. Five weeks after inoculation C. polonica had penetrated phloem and sapwood further than any other fungus. It grew slower than the other fungi in both tissues the first week after inoculation, but the four following weeks it grew faster than all other fungi.

Abstract

Aggressive bark beetles kill healthy conifers through pheromone-mediated mass attacks. The exact mechanism by which trees are killed is still disputed, but phytopathogenic blue-stain fungi associated with the beetles are probably involved in most cases. This thesis compares the blue-stain flora of five bark beetle species that colonize Norway spruce of different resistance. The aggressive Ips typographus can kill healthy trees, while the other species are non-aggressive and colonize either severly stressed or dying trees (the facultatively parasitic Ips duplicatus, Polygraphus poligraphus and Pityogenes chalcographus), or dead trees (the saprophagic Hylurgops palliates). Ips typographus and I. duplicatus were both found to carry very high frequencies of the phytopathogenic fungus Ceratocystis polonica. This fungus has previously been found associated only with I. typographus, and has been shown to be pathogenic to Norway spruce in experimental mass inoculations. The other bark beetles carried no known pathogenic fungi. The phytopathogenicity of four of the isolated blue-stain fungi were evaluated through mass and low-density inoculation in young Norway spruce trees. Mass inoculations confirmed C. polonica\"s pathogenicity, while the other fungi, which are associated with non-aggressive beetles, were not pathogenic (Ophiostoma piceae, cfr. Ambrosiell sp., Dark sterile sp.A). Low-density inoculations revealed only small differences between the four fungi in phloem necrosis length. Necrosis length is used as a standard criterion of fungal pathogenicity, but it was not useful for assessing the pathogenicity of the fungi in the fungus-host tree system under study here. Ceratocystis polonica was found to penertrate deeper into the sapwood and induce deeper sapwood desiccation than the other fungi. The ability to invade sapwood is probably more important for fungal pathogenicity than the ability to colonize phloem, and may thus be a better criterion for assessing the pathogenicity of blue-stain fungi. General aspects of the association between bark beetles and blue-stain fungi are discussed.