To Örebro University

Small 5 to 7 cm seedlings appear to be less damaged by pine weevils (Hylobius abietis L.), one of the most economically devastating forest pests in Europe. In attempts to understand the underlying mechanisms, volatiles (VOC) released by the whole plant and wounded phloem of Scots pine (P. sylvestris L.) seedlings were investigated during the first year of growth. The changes in the VOC due to age/developmental stage and storage regimes were determined. Majority of the seedlings (84%) represented a '3-carene chemotype' and 15% an 'alpha-pinene chemotype'. 3.5 Week-old intact or wounded seedlings emitted mainly geranyl acetone and green leaf volatiles. Proportional increase of (+)-3-carene occurred during the growth and aging of intact '3-carene seedlings', while (+/-)-alpha-pinene changed in their phloem emissions. From 9.5 week onwards, the intact seedlings began to emit 2-phenylethanol. During winter dormancy, the pine odor enriched with estragole, 3-phenylpropanol and 4-ethyl-2-methoxyphenol. The latter one, together with 4-ethylphenol, was characteristic to the over-wintered in an outdoor area (OA). These results support the hypothesis that seedling attractiveness to herbivores depend on the plant age and overwintering practices. Considering the antimicrobial properties of volatiles, overwintering of plants under natural conditions may also ensure higher disease resistance of plants.

Outbreaks of the Eurasian spruce bark beetle (Ips typographus) have decimated millions of hectares of conifer forests in Europe in recent years. The ability of these 4.0 to 5.5 mm long insects to kill mature trees over a short period has been sometimes ascribed to two main factors: (1) mass attacks on the host tree to overcome tree defenses and (2) the presence of fungal symbionts that support successful beetle development in the tree. While the role of pheromones in coordinating mass attacks has been well studied, the role of chemical communication in maintaining the fungal symbiosis is poorly understood. Previous evidence indicates that I. typographus can distinguish fungal symbionts of the genera Grosmannia, Endoconidiophora, and Ophiostoma by their de novo synthesized volatile compounds. Here, we hypothesize that the fungal symbionts of this bark beetle species metabolize spruce resin monoterpenes of the beetle's host tree, Norway spruce (Picea abies), and that the volatile products are used as cues by beetles for locating breeding sites with beneficial symbionts. We show that Grosmannia penicillata and other fungal symbionts alter the profile of spruce bark volatiles by converting the major monoterpenes into an attractive blend of oxygenated derivatives. Bornyl acetate was metabolized to camphor, and α- and β-pinene to trans-4-thujanol and other oxygenated products. Electrophysiological measurements showed that I. typographus possesses dedicated olfactory sensory neurons for oxygenated metabolites. Both camphor and trans-4-thujanol attracted beetles at specific doses in walking olfactometer experiments, and the presence of symbiotic fungi enhanced attraction of females to pheromones. Another co-occurring nonbeneficial fungus (Trichoderma sp.) also produced oxygenated monoterpenes, but these were not attractive to I. typographus. Finally, we show that colonization of fungal symbionts on spruce bark diet stimulated beetles to make tunnels into the diet. Collectively, our study suggests that the blends of oxygenated metabolites of conifer monoterpenes produced by fungal symbionts are used by walking bark beetles as attractive or repellent cues to locate breeding or feeding sites containing beneficial microbial symbionts. The oxygenated metabolites may aid beetles in assessing the presence of the fungus, the defense status of the host tree and the density of conspecifics at potential feeding and breeding sites.

An essential component of plant defense is the change that occurs from a constitutive to an induced state following damage or infection. Exogenous application of the plant hormone methyl jasmonate (MeJA) has shown great potential to be used as a defense inducer prior to pest exposure, and could be used as a plant protection measure. Here, we examined (1) the importance of MeJA-mediated induction for Norway spruce (Picea abies) resistance against damage by the pine weevil Hylobius abietis, which poses a threat to seedling survival, and infection by the spruce bark beetle-associated blue-stain fungus Endoconidiophora polonica, (2) genotypic variation in MeJA-induced defense (terpene chemistry), and (3) correlations among resistance to each pest. In a semi-field experiment, we exposed rooted-cuttings from nine different Norway spruce clones to insect damage and fungal infection separately. Plants were treated with 0, 25, or 50 mM MeJA, and planted in blocks where only pine weevils were released, or in a separate block in which plants were fungus-inoculated or not (control group). As measures of resistance, stem area debarked and fungal lesion lengths were assessed, and as a measure of defensive capacity, terpene chemistry was examined. We found that MeJA treatment increased resistance to H. abietis and E. polonica, but effects varied with clone. Norway spruce clones that exhibited high constitutive resistance did not show large changes in area debarked or lesion length when MeJA-treated, and vice versa. Moreover, insect damage negatively correlated with fungal infection. Clones receiving little pine weevil damage experienced larger lesion lengths, and vice versa, both in the constitutive and induced states. Changes in absolute terpene concentrations occurred with MeJA treatment (but not on proportional terpene concentrations), however, variation in chemistry was mostly explained by differences between clones. We conclude that MeJA can enhance protection against H. abietis and E. polonica, but the extent of protection will depend on the importance of constitutive and induced resistance for the Norway spruce clone in question. Trade-offs among resistances do not necessarily hinder the use of MeJA, as clones that are constitutively more resistant to either pest, should show greater MeJA-induced resistance against the other. 

Constitutive and inducible terpene production is involved in conifer resistance against insects and fungal infestations. To gain knowledge about local defence responses of Norway spruce bark against pathogens and to find potential chemical markers for resistance breeding, we inoculated the stem of 8-year-old Norway spruce (Picea abies) clonal trees with bothEndoconidiophora polonica(Ep, a common fungal pathogen associated with the spruce bark beetleIps typographus) andHeterobasidion parviporum(Hp, a severe pathogen causing root and stem rot disease). Three weeks after inoculation, the fungal-inoculated and intact bark from each tree was sampled. The terpenes in tree bark were extracted with hexane and characterized by gas chromatography-mass spectrometry (GC-MS). The two fungi induced varied terpene responses in the four spruce clones used. Three of the clones showed a 2.3-fold to 5.7-fold stronger terpene response to Hp relative to Ep inoculation, while one clone responded similarly to inoculation with the two fungal pathogens. The amount of the diterpenes thunbergol and geranyllinalool varied between the clones. The level of thunbergol was higher in both intact and fungal-inoculated bark from the less susceptible clones compared with the more susceptible clones. Geranyllinalool was present in higher amounts in the susceptible clones and is thus a possible marker for susceptibility. Our observations show that Norway spruce employs a similar chemical mechanism against the two fungal pathogens. Based on the present and earlier published data, we suggest that certain Norway spruce genotypes have a strong defence reaction against these two pathogens. The diterpenes thunbergol and geranyllinalool might be useful markers of susceptibility in tree-breeding programmes and should be the focus of further detailed investigations.

In response to various stimuli, plants acquire resistance against pests and/or pathogens. Such acquired or induced resistance allows plants to rapidly adapt to their environment. Spraying the bark of mature Norway spruce (Picea abies) trees with the phytohormone methyl jasmonate (MeJA) enhances resistance to tree-killing bark beetles and their associated phytopathogenic fungi. Analysis of spruce chemical defenses and beetle colonization success suggests that MeJA treatment both directly induces immune responses and primes inducible defenses for a faster and stronger response to subsequent beetle attack. We used metabolite and transcriptome profiling to explore the mechanisms underlying MeJA-induced resistance in Norway spruce. We demonstrated that MeJA treatment caused substantial changes in the bark transcriptional response to a triggering stress (mechanical wounding). Profiling of mRNA expression showed a suite of spruce inducible defenses are primed following MeJA treatment. Although monoterpenes and diterpene resin acids increased more rapidly after wounding in MeJA-treated than control bark, expression of their biosynthesis genes did not. We suggest that priming of inducible defenses is part of a complex mixture of defense responses that underpins the increased resistance against bark beetle colonization observed in Norway spruce. This study provides the most detailed insights yet into the mechanisms underlying induced resistance in a long-lived gymnosperm. 

Ophiostomatalean fungi may facilitate bark beetle colonization and reproduction. In the present study of the fungal community associated with bark beetle spe-cies belonging to Tomicus in Yunnan, China, six ophios-tomatalean fungi (Ophiostoma canum, O. ips, O. tingens, Leptographium yunnanense, Leptographium sp. 1 and Lep-tographium sp. 2) were isolated from the beetles or their galleries; O. canum was the most common fungal species. The distribution of O. canum was associated with stands heavily damaged by Tomicus species and a higher percentage of valid galleries of Tomicus yunnanensis and T. minor in Yunnan pine (Pinus yunnanensis). After inoculation of Yun-nan pine with the fungus, a phloem reaction zone formed and monoterpenes accumulated in the phloem. These results suggested that O. canum was pathogenic to Yunnan pine and that the wide distribution of the fungus might be beneficial to reproduction of pine shoot beetles in Yunnan pine. How-ever, because the reaction zone and monoterpene accumu-lation were mild, fungal damage of Yunnan pine might be limited. A more integrated study considering all the fungal species should be done to better understand the interactions among bark beetles, blue-stain fungi, and the tree hosts in the region.

Plants can form an immunological memory known as defense priming, whereby exposure to a priming stimulus enables quicker or stronger response to subsequent attack by pests and pathogens. Such priming of inducible defenses provides increased protection and reduces allocation costs of defense. Defense priming has been widely studied for short–lived model plants such as Arabidopsis, but little is known about this phenomenon in long‐lived plants like spruce. We compared the effects of pre‐treatment with sub‐lethal fungal inoculations or application of the phytohormone methyl jasmonate (MeJA) on the resistance of 48‐year‐old Norway spruce (Picea abies) trees to mass attack by a tree‐killing bark beetle beginning 35 days later. Bark beetles heavily infested and killed untreated trees, but largely avoided fungus‐inoculated trees and MeJA‐treated trees. Quantification of defensive terpenes at the time of bark beetle attack showed fungal inoculation induced 91‐fold higher terpene concentrations compared to untreated trees, while application of MeJA did not significantly increase terpenes. These results indicate that resistance in fungus‐inoculated trees is a result of direct induction of defenses while resistance in MeJA‐treated trees is due to defense priming. This work extends our knowledge of defense priming from model plants to an ecologically important tree species.

Emissions of defence chemicals from Norway spruce seedlings can be induced by feeding arthropods or by exogenous hormonal application. Some defence chemicals may attract or repel associated arthropods. The aim of this study was to show that it is possible to detect and collect stress-induced volatiles from micro sites, such as at the scale of a single needle, in vivo by using SPME. Methyl jasmonate application on the stem of Norway spruce seedlings induced emission of (E)-beta-farnesene only from the needles closest to the application site. Emissions of (E)-beta-farnesene, (E,E)-alpha-farnesene and (E)-alpha-bisabolene were only detected from needles infested by the spider mite Oligonychus ununguis. The total volatile amount detected by SPME-GC-MS reached a considerable mass of 14 ng/needle/24 h, suggesting that emission from damaged and stressed conifers might have a larger impact on the macro climate than previously estimated.

Convergent evolution of semiochemical use in organisms from different Kingdoms is a rarely described phenomenon. Tree-killing bark beetles vector numerous symbiotic blue-stain fungi that help the beetles colonize healthy trees. Here we show for the first time that some of these fungi are able to biosynthesize bicyclic ketals that are pheromones and other semiochemicals of bark beetles. Volatile emissions of five common bark beetle symbionts were investigated by gas chromatography-mass spectrometry. When grown on fresh Norway spruce bark the fungi emitted three well-known bark beetle aggregation pheromones and semiochemicals (exo-brevicomin, endo-brevicomin and trans-conophthorin) and two structurally related semiochemical candidates (exo-1,3-dimethyl-2,9-dioxabicyclo[3.3.1]nonane and endo-1,3-dimethyl-2,9-dioxabicyclo[3.3.1]nonane) that elicited electroantennogram responses in the spruce bark beetle Ips typographus. When grown on malt agar with C-13 D-Glucose, the fungus Grosmannia europhioides incorporated C-13 into exo-brevicomin and trans-conophthorin. The enantiomeric compositions of the fungus-produced ketals closely matched those previously reported from bark beetles. The production of structurally complex bark beetle pheromones by symbiotic fungi indicates cross-kingdom convergent evolution of signal use in this system. This signaling is susceptible to disruption, providing potential new targets for pest control in conifer forests and plantations.

The bark beetle Ips typographus carries numerous fungi that could be assisting the beetle in colonizing live Norway spruce (Picea abies) trees. Phenolic defenses in spruce phloem are degraded by the beetle's major tree-killing fungus Endoconidiophora polonica, but it is unknown if other beetle associates can also catabolize these compounds. We compared the ability of five fungi commonly associated with I. typographus to degrade phenolic compounds in Norway spruce phloem. Grosmannia penicillata and Grosmannia europhioides were able to degrade stilbenes and flavonoids faster than E. polonica and grow on minimal growth medium with spruce bark constituents as the only nutrients. Furthermore, beetles avoided medium amended with phenolics but marginally preferred medium colonized by fungi. Taken together our results show that different bark beetle-associated fungi have complementary roles in degrading host metabolites and thus might improve this insect's persistence in well defended host tissues.