Priming of inducible defenses protects Norway spruce against tree-killing bark beetles.

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 pretreatment with sublethal 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 with untreated trees, whereas 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, whereas 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.

Random and Directed Movement by Warren Root Collar Weevils (Coleoptera: Curculionidae), Relative to Size and Distance of Host Lodgepole Pine Trees.

Hylobius warreni Wood (Coleoptera: Curculionidae) is a pest of conifers, especially lodgepole pine (Pinus contorta var. latifolia Douglas ex Loudon) (Pinales: Pinaceae) in the Interior of British Columbia. The larvae feed on the roots and root collars and cause girdling damage, resulting in mortality or growth reductions. Previous research has suggested the adult weevils locate potential host trees by using random movements and vision, but likely not chemosensory cues. The purpose of this study is to determine if adult H. warreni respond to particular tree characteristics versus encounter potential hosts at random. Study A was a capture-mark-recapture experiment where weevils were captured on mature pine trees, while Study B was a tracking experiment within a young pine plantation. Weevils showed a preference for larger trees, and for trees that were closer to the weevil's last known location. In Study A, weevils also avoided climbing trees in poor health, while in Study B, the weevils' preference for taller trees increased as their distance from the weevil increased, as well as when taller trees were closer to other trees. Movement rates were similar to those observed in previous studies, were positively correlated with the average spacing of trees, and declined with time after release. This confirms previous findings that H. warreni may locate host trees by both vision and random movements, and that their movements are determined primarily by the size and distribution of potential host trees within their habitat.

Comparison of different site preparation techniques: quality of planting spots, seedling growth and pine weevil damage.

In northern Europe, there are high risks of severe pine weevil (Hylobius abietis) damage to newly planted conifer seedlings. Site preparation is one of the most important measures for reducing these risks and as several studies have shown the damage is highly dependent on the amount of pure mineral soil around the seedlings. We investigated effects of three site preparation techniques: (1) disc trenching with a conventional Bracke T26, (2) MidiFlex unit and (3) soil inversion with a Karl Oskar unit on characteristics of the planting spots, growth and pine weevil damage and survival rates of untreated and insecticide treated planted Norway spruce (Picea abies) seedlings. All three site preparation techniques reduced pine weevil damage in comparison with no site preparation, and the proportion of spots with pure mineral soil they created was inversely related to the rate of mortality caused by pine weevil. The results indicate that the quality of the planting spots depends on the technique used. In areas where pine weevil is the major threat to seedling survival, the amount of mineral soil in the planting spots is the most important factor in order to protect the seedling from damage. Without site preparation most planting spots consisted of undisturbed humus. Generally, the Karl-Oskar created the most spots with pure mineral soil, but on very stony soils the Bracke T26 created more mineral soil spots than other methods. Site preparation is a valuable tool in order to improve survival in regeneration areas and it is of great importance to make the right choice of technique depending on the particular circumstances on the actual site.

Methyl Jasmonate-Induced Monoterpenes in Scots Pine and Norway Spruce Tissues Affect Pine Weevil Orientation.

In large parts of Europe, insecticide-free measures for protecting conifer plants are desired to suppress damage by the pine weevil Hylobius abietis (L.). Treatment with methyl jasmonate (MeJA), a chemical elicitor already used in crop production, may enhance expression of chemical defenses in seedlings in conifer regenerations. However, in a previous experiment, MeJA treatment resulted in substantially better field protection for Scots pine (Pinus sylvestris L.) than for Norway spruce (Picea abies (L.) Karst.). Hypothesizing that the variations may be at least due partly to volatiles released by MeJA-treated seedlings and their effects on pine weevil orientation, we examined tissue extracts of seedlings (from the same batches as previously used) by two-dimensional GC-MS. We found that the MeJA treatment increased contents of the monoterpene (-)-ß-pinene in phloem (the weevil's main target tissue) of both tree species, however, the (-)-ß-pinene/(-)-α-pinene ratio increased more in the phloem of P. sylvestris. We also tested the attractiveness of individual monoterpenes found in conifer tissues (needles and phloem) for pine weevils using an arena with traps baited with single-substance dispensers and pine twigs. Trap catches were reduced when the pine material was combined with a dispenser releasing (-)-ß-pinene, (+)-3-carene, (-)-bornyl acetate or 1,8-cineole. However, (-)-α-pinene did not have this effect. Thus, the greater field protection of MeJA-treated P. sylvestris seedlings may be due to the selective induction of increases in contents of the deterrent (-)-ß-pinene, in contrast to strong increases in both non-deterrent (-)-α-pinene and the deterrent (-)-ß-pinene in P. abies seedlings.

Variation in Methyl Jasmonate-Induced Defense Among Norway Spruce Clones and Trade-Offs in Resistance Against a Fungal and an Insect Pest.

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.

Novel Avenues for Plant Protection: Plant Propagation by Somatic Embryogenesis Enhances Resistance to Insect Feeding.

Somatic embryogenesis (SE), a clonal propagation method utilizing somatic cells, occurs under conditions that activate plant stress adaptation mechanisms such as production of protective secondary metabolites. Surprisingly, possible differences in susceptibility to insect pests between SE-generated and conventionally cultivated plants have not been previously explored. Here, we recorded frequencies and levels of bark-feeding damage by pine weevils (Hylobius abietis) in two large field trials, consisting of emblings (SE-propagated plants) and seedlings from 50 half-sib Norway spruce (Picea abies) families. We found that emblings were less frequently attacked by pine weevils, and when attacked, they were damaged to a lesser extent than seedlings. Moreover, we detected significant additive genetic variation in damage levels received by plants, indicating a heritable component to differences in resistance to insect herbivory among half-sib families. We present first-time evidence that emblings can be more resistant than seedlings to herbivorous insect damage, thus, SE appears to confer a previously unknown plant protection advantage. This finding indicates novel avenues to explore mechanisms underlying plant resistance and new approaches to develop non-toxic measures against insect pests.

Pest risk assessment of Eotetranychus lewisi for the EU territory.

Following the 2014 EFSA's Panel on Plant Health scientific opinion on the pest categorisation of the spider mite Eotetranychus lewisi, the European Commission requested the Panel to perform a pest risk assessment and evaluate the risk reduction options. A stochastic model was used to assess entry, establishment and spread and related uncertainties. In the EU, E. lewisi has only been reported to occur in Portugal (Madeira). Entry pathways assessed were strawberry plants for planting from the USA, poinsettia and raspberry plants for planting, and orange and lemon fruits from third countries. Entry is most likely via poinsettia. Under current EU phytosanitary requirements, there is around a one in ten chance that E. lewisi will establish outdoors over the next 10 years. Although unlikely, establishment would most likely occur in southern Europe where environmental conditions, temperature and host density, are most suitable. If E. lewisi did establish, pest spread is expected to be mainly human assisted, most likely the mite being transported long distances on plants for planting. Nevertheless, while remaining a regulated pest, spread would be slow and most likely confined to one NUTS 2 area after 10 years. Under a scenario with enhanced measures (pest free place of production) at origin, the Panel's assessment indicate that it is extremely unlikely that E. lewisi would establish within 10 years hence spread is also extremely unlikely. The absence of trade of host plants from Madeira to other parts of the EU could explain why E. lewisi has not spread to other EU Member States. E. lewisi is reported as reducing yield and quality of peaches and poinsettia and is regarded as a growing concern for strawberry and raspberry growers in the Americas. The Panel concludes that should E. lewisi be introduced in the EU similar impacts could be expected.

Induced defenses change the chemical composition of pine seedlings and influence meal properties of the pine weevil Hylobius abietis.

The defense of conifers against phytophagous insects relies to a large extent on induced chemical defenses. However, it is not clear how induced changes in chemical composition influence the meal properties of phytophagous insects (and thus damage rates). The defense can be induced experimentally with methyl jasmonate (MeJA), which is a substance that is produced naturally when a plant is attacked. Here we used MeJA to investigate how the volatile contents of Scots pine (Pinus sylvestris L.) tissues influence the meal properties of the pine weevil (Hylobius abietis (L.)). Phloem and needles (both weevil target tissues) from MeJA-treated and control seedlings were extracted by n-hexane and analyzed by two-dimensional gas chromatography-mass spectrometry (2D GC-MS). The feeding of pine weevils on MeJA-treated and control seedlings were video-recorded to determine meal properties. Multivariate statistical analyses showed that phloem and needle contents of MeJA-treated seedlings had different volatile compositions compared to control seedlings. Levels of the pine weevil attractant (+)-α-pinene were particularly high in phloem of control seedlings with feeding damage. The antifeedant substance 2-phenylethanol occurred at higher levels in the phloem of MeJA-treated than in control seedlings. Accordingly, pine weevils fed slower and had shorter meals on MeJA-seedlings. The chemical compositions of phloem and needle tissues were clearly different in control seedlings but not in the MeJA-treated seedlings. Consequently, meal durations of mixed meals, i.e. both needles and phloem, were longer than phloem meals on control seedlings, while meal durations on MeJA seedlings did not differ between these meal contents. The meal duration influences the risk of girdling and plant death. Thus our results suggest a mechanism by which MeJA treatment may protect conifer seedlings against pine weevils.

Induced terpene accumulation in Norway spruce inhibits bark beetle colonization in a dose-dependent manner.

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 m(-2)) and 2.6% as much gallery length (0.029 m m(-2) vs. 1.11 m m(-2)) 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 g(-1) dry phloem trees suffered only moderate beetle colonization, and above a high threshold of ∼200 mg terpene g(-1) 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.

Seeing the forest through the trees: differential dispersal of Hylobius warreni within modified forest habitats.

Hylobius warreni Wood, also known as the Warren root collar weevil, is a flightless insect that feeds on conifers throughout the boreal forests of Canada. Mature trees typically can withstand feeding, but larval feeding around the root collar may cause mortality to young trees. Recently, a large outbreak of mountain pine beetle (Dendroctonus ponderosae Hopkins) has killed a high proportion of mature lodgepole pine (Pinus contorta Douglas variety latifolia) across British Columbia, Canada. This raises concerns that adult weevils may migrate from mature forests with reduced host pools into adjacent young forests that had been salvaged and replanted. To study movement of these walking weevils in different habitat types, we constructed three research plots consisting of various combinations of live-, dead-, and mixed (i.e., live and dead)-tree habitats. We observed dispersal patterns of individually labeled insects using a novel insect trap attached to the base of trees. Approximately 35% of insects were recaptured over 1 mo. Weevils were least likely to be recaptured proximate to the release location when released in a habitat with dead trees. Movement rates therein were almost double the rates of insects moving through live- or mixed-tree habitats. Our findings support the hypothesis that H. warreni may disperse out of habitats with dead trees into areas with higher proportions of green trees. Our findings are discussed in the context of habitat discrimination and potential increases in herbivory by H. warreni in western Canada given salvage harvesting activities after outbreaks of mountain pine beetle.

The influence of Ceratocystis polonica inoculation and methyl jasmonate application on terpene chemistry of Norway spruce, Picea abies.

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 (+)-alpha-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 (-)-beta-pinene and (-)-limonene. Fungal inoculation significantly increased the enantiomeric ratio of (-)-alpha-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 (-)-beta-pinene, (-)-beta-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 (-)-alpha-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.