Protection of spruce seedlings against pine weevil attacks by treatment of seeds or seedlings with nicotinamide, nicotinic acid and jasmonic acid

here Norway spruce ( Picea abies (L.) Karst.) seeds were treated with 2.5 mM nicotinamide (NIC), 2.5 mM nicotinic acid(NIA),3 mMjasmonicacid(JA)or0.2 mM5-azacytidine(5-Aza),and6-month-oldseedlingsgrownfromthese seeds wereplantedatareforestationareain centralSweden.Attackbypineweevils( Hylobiusabietis )wasreduced by 50 per cent by NIC treatment, 62.5 per cent by JA treatment and 25 per cent by 5-Aza treatment, when compared with seedlings grown from untreated seeds. Watering 18-month-old spruce seedlings with 2 mM NIC or 2 mM NIA did reduce attack during the ﬁrst season in the ﬁeld by 40 and 53 per cent, respectively, compared with untreated plants. Girdling was also reduced by the different treatments. Analysis of conifer seedlings treated with 5-Aza points at a possible involvement of epigenetic mechanisms in this defensive capacity. This is supported bya reduced level of DNA methylation in the needles of young spruce seedlings grown in a greenhouse from NIC-treated seeds. Seed treatment for seedling defense potentiation is simple, inexpensive and also a new approach for forestry with many potential applications.


Introduction
Forestry resources in Northern European countries are commonly replenished by reforestation, where new seedlings are planted soon after clearcutting of an older area of forest. In 2012, this method was utilized on 73 per cent of the regenerated forestry area in Sweden. This required a total of 374 million planted seedlings, of which spruce (Picea abies (L.) Karst.) and pine (Pinus sylvestris L.) were the dominating species, representing 58 and 34 per cent of new plantings, respectively (Christiansen, 2013). The large pine weevil (Hylobius abietis L., Coleoptera: Curculionidae) is known to cause severe damage to unprotected young conifer seedlings planted after clearcutting, by gnawing on the stem bark of the newly planted seedlings (Lå ngströ m and Day, 2004). Traditionally, the protection strategy of choice has been the application of insect neurotoxic pesticides, primarily pyrethroids, applied by spraying or dipping seedlings (Lå ngströ m and Day, 2004 and references therein). However, these chemicals have a toxicological impact on the environment, especially on waterdwelling organisms (Mian and Mulla, 1992), due to leakage into soil and ground water. These insecticides may also be hazardous to the humans producing and handling the compounds (Kolmodin-Hedman et al., 1995). Pyrethroid-type pesticides are therefore no longer in use in forestry, although similar compounds are still permitted, including the lambdacyhalotrin-based cypermethrin and imidacloprid. Exemptions permitting the use of imidacloprid are sometimes given to companies that are certified by the Forest Stewardship Council (FSC), which are otherwise not permitted to use any pesticides. Various alternatives to these toxicological chemical treatments have been proposed; most are physical approaches to prevent pine weevils from attacking seedlings, such as coating stems with wax (Petersson et al., 2004) or sand (Nordlander et al., 2011). These physical methods can be effective, but are labor-intensive and much more expensive than broad application of conventional insecticides (Lå ngströ m and Day, 2004).
Other ecological alternatives have also been proposed, including the application of antifeedant substances which can be found in the feces of the pine weevil (Unelius et al., 2006), and the planting of so-called mini seedlings (Lindströ m et al., 2005;Danielsson et al., 2008). There are also several silvicultural measures that can be taken which reduce pine weevil population and therefore also reduce the need for seedling protection. These measures involve the use of scarification, planting in mineral soil, allowing a fallow period before planting and planting seedlings beneath larger shelter trees (Lå ngströ m and Day, 2004;Nordlander et al., 2011). These approaches all have promise in protecting seedlings from insect attack. However, as long as broad clearcutting remains the dominant approach to forest regeneration, there will always be a need for the protection of individual seedlings from insect attack, as this cultivation method favours the development of large weevil populations on regeneration sites.
One promising new approach to seedling protection is the use of chemical elicitors, such as the well-known plant defense signalling compound methyl jasmonate (MeJA), which has been used in attempts to improve conifer seedling resistance to pine weevils (Heijari et al., 2005;Holopainen et al., 2009;Zas et al., 2014). Exogenous application of such chemicals induces the plant's natural defensive capabilities, without introducing toxic new compounds to the ecosystem. The application method has primarily been by spraying or fumigation. Trials of MeJA-sprayed conifers indicate that the presence of stem resin(s) is an important feature of defense against pine weevil attack , but little is generally known about the precise effect of other chemical elicitors on conifer defense against pine weevils. In this context, the defense-activating compound nicotinamide (NIC) (Berglund, 1994), also known as niacinamide, and its metabolite in plants, nicotinic acid (NIA), better known as vitamin B 3 (niacin), have here been tested for their ability to improve the defensive capacity of young spruce seedlings to pine weevil attack. NIC is known to influence a plethora of defensive activities in both animal (Surjana et al., 2010;Canto et al., 2013) and plant cells (Berglund et al., 1993a,b;Berglund, 1994;Ohlsson et al., 2008). NIC, and its plant metabolite NIA, have also been suggested to function as stress signal mediating compounds in eukaryotic cells (Berglund, 1994). Furthermore, isonicotinamide (Basson and Dubery, 2007) and 2,6-dichloro-isonicotinic acid (Metraux et al., 1991), synthetic analogues of NIC and NIA, respectively, have been used to induce defense in various non-conifer plant species, which supports a role for the naturally occurring compounds NIC and NIA in native plant defense. Previous results indicated that NIC acts at a general level in plants as well as in animals, as various defense pathways and processes are activated, reflected in changes to gene expression patterns (Berglund and Ohlsson 1995;Surjana et al., 2010).
Induced gene expression in plants and other eukaryotes depends on at least two factors: an endogenous or exogenous molecular signal is needed, and DNA must be accessible for interaction with the signal. In eukaryotic cells, DNA is packed together with proteins (histones) into a structure called chromatin, which must be unpacked to be available for interactions with other molecules. This process is influenced by features such as the level of DNA methylation and various histone modifications, which contribute to the so-called epigenetic regulatory mechanisms of gene expression, which can in turn be influenced by environmental factors and cellular signals (Cedar and Bergman, 2009;Brä utigam et al., 2013;Kinoshita and Seki, 2014). Thus, it is not just the level of intrinsic or extrinsic inducing signals that determines the response, but also the state of chromatin packing. In the present study 5-azacytidine (5-Aza), a well-known inhibitor of DNA methylation (Yang et al., 2010), was used as a reference substance for investigation of the possible influence of DNA methylation in defense activation.
Priming, also known as sensitization, is a strategy by which plants can accelerate and perhaps potentiate a defensive response when later exposed to a second occurrence of a certain kind of stress (Pastor et al., 2013). This is an energy-saving mechanism which allows plants to mount a timely defensive strategy to biotic or abiotic stresses without the wasteful and unnecessary constitutive production of defensive molecules like proteins and secondary metabolites. The mechanisms behind priming are not well-known, but may involve increased levels of active transcription factors, as well as unidentified epigenetic mechanisms (Pastor et al., 2013).
It is well-known that seed treatment can influence the performance of seedlings or mature plants within agriculture. For example, jasmonic acid (JA) treatments of seeds from tomato plants led to mature plants with a strong defensive capability against attack by arthropod herbivores and fungal pathogens (Worrall et al., 2012). As far as we know, seed treatment has not so far been reported to promote insect defense in conifers, although it has been shown that spruce embryos treated by changes in temperature, sensitizing them to environmental fluctuations, can influence the ability of spruce plants to handle some abiotic parameters (Yakovlev et al., 2011). We hypothesize that it is possible to potentiate spruce seedling defense against pine weevils via a short seed exposure or via seedling watering with defense potentiating compounds, and that epigenetic mechanisms are involved in this defense potentiation. The research questions in this study were: (1) can spruce treatment with the plant defense potentiating compounds NIC or NIA promote defense against pine weevil attack? (2) Can JA seed treatment give the mature plants protection against pine weevils? and (3) can treatment of spruce seeds with compounds known to generally decrease DNA methylation influence a plant's defense against pine weevils? It was hypothesized that these treatments would prime the seeds or seedlings, rendering the fully grown plants more capable of mounting a swift defensive response to signals arising from environmental stresses, primarily including insect attack.

Plant material
All seeds utilized in this study were Norway spruce seeds, origin 578 00 ′ N, altitude 55 m, collected in the orchard of Ö hn. The treatments described below were carried out either on these seeds or on seedlings grown from them. Seedlings were grown in various container types, all filled with Finnish peat (Kekkilä Oy, Tuusula, Finland). A complete mineral solution was used to fertilize the growing seedlings (Wallco, Sweden: N:P:K, 100:13:65 w/v).

Experiment with seed treated seedlings
Seeds were treated on 18 April 2010 with test substances in water solutions under gentle shaking for 4 h at 238C in darkness. The test substances were NIC (2.5 mM), NIA (2.5 mM), JA (3 mM) and 5-Aza (200 mM). The surfactant Tween 80 (0.24 ml ml 21 ) was added to the solutions to increase the contact between the substances and the seeds. To rule out the potential influence of the surfactant, in this study the control seeds were treated with water containing Tween 80, as defense-inducing effects have been observed for Tween (Moreira et al., 2009). After treatment, seeds were allowed to dry on filter paper overnight before sowing. The following day 90 ml containers (Hiko V 90, BCC, Sweden) filled with peat were seeded with treated seeds at the research station in Vassbo (608 32 ′ N; 158 33 ′ E). Before sowing, the container units were split in half, resulting in 20 cavities per container unit. Each container was then sown with two seeds from each of the five treatments. In total, four container units (replicates) per seed treatment were sown. After sowing, the units were arranged in a completely randomized (CR) design in the greenhouse. During germination the relative humidity Forestry was kept at 70 per cent and the temperature at an average of 208 C. After 9 days, the germination results for the different treatments were investigated visually by assessing how far the germ had developed. Fertilization started the third week after sowing with a weekly nitrogen supply of 3 g N m 22 . After 3 weeks the seedlings were thinned so that only one seedling per container was left. Seedlings were kept in the greenhouse until the middle of June, when they were put outdoors for further growth.
On 20 August, the spruce seedlings were planted at Kann-Olles Heden (608 38 ′ N; 168 13 ′ E). This regeneration site was clear cut in 2008 and scarified with a harrow during late spring 2010. The site index is G 24, as 24 m is the dominant height of Norway spruce at the age of 100 years according to Hä gglund and Lundmark (1982), and the soil type at the site is a sandyloam till. In total, 200 spruce seedlings were planted in a CR-design with 4 replicate sets of 10 seedlings for each of the 5 treatments. Where possible, the seedlings were placed in the mineral soil, avoiding humus and the deeper parts of the harrow furrows. The heights of the seedlings were recorded when they were planted, while seedling vitality and the extent of pine weevil gnawing were registered for each seedling (gnawed bark area cm 2 ) on 18 October by visual assessment.

Experiment with watering
Untreated spruce seeds were sown in July 2009 in a commercial nursery, Nä ssja nursery (608 15 ′ N; 168 50 ′ E), in 15 ml mini containers, and grew to seedlings which were to be treated via watering. In the middle of April 2010 the mini seedlings were collected and transported to the research station in Vassbo and immediately transplanted into larger 85 ml containers (Plantek 81, BCC, Sweden) for further growth. Before transplanting, the containers were filled with a peat growing medium. Three container units each containing 81 seedlings were randomly selected for one of three treatments: water (control), NIC and NIA. The container units were then randomly positioned for growth in a CR design. Seedlings were treated by watering (2 l m 22 ) twice a week with 2 mM NIC or NIA dissolved in the water from the middle of April until the end of June. The water also contained a dissolved complete mineral nutrient solution, and the weekly nitrogen supply was 3 g N m 22 . Seedlings were grown in a greenhouse at an average constant temperature of 208 C until the middle of June when they were put outdoors. Just before out-planting in the field, at the end of June, 30 seedlings from each of the treatments were randomly selected for measuring of height, stem diameter and root and shoot dry weight. Seedlings were then planted at Gettjä rnsberget (608 30 ′ N; 168 02 ′ E), which had been clearcut in the autumn of 2008 and scarified with a harrow in the autumn of 2009. The site index is G 24 and the soil type is a sandy-loam till. The seedlings were planted in five randomized blocks, each containing three plots with 11 seedlings of each treatment. In early October, the seedlings were examined with respect to vitality and damage caused by the pine weevil.

Combination of seed treatment and watering with NIC for laboratory tests
Seeds were treated, and seedlings cultivated, as described above. To test the effect of a second exposure to NIC, seedlings grown from seeds treated with NICwere watered with NIC after 19 weeks of growth. Seedlings were watered to 2 l m 22 twice a week with 2 mM NIC. This treatment lasted for 3 weeks. Tests with pine weevils and seedlings were carried out in the laboratory.
Six seedlings of each of the two treatments, the control (untreated) and the twice NIC-treated seedlings (seedlings grown from NIC-treated seeds and subsequently watered with NIC), were planted in plastic containers. The containers with seedlings were randomly lowered down through holes in the bottom of a rectangular box so that the soil surface was in line with the bottom of the box. The box had internal measurements of 1.0×0.7 and 0.2 m high walls, which were painted with fluon on the inside of the box to prevent pine weevils from climbing and escaping.
The top of the box was covered with a net for additional protection. The positioning of seedlings in the box was according to a CR design. Two tests were performed with 20 pine weevils placed in the box when starting the tests. The first test went on for 48 h, after which seedlings were examined for bark gnawing and the gnawed area of the seedlings was estimated. The first test was repeated with new seedlings and ran for 60 h.

DNA methylation
For analysis of DNA methylation, seed treatment and seedling cultivation were performed as described above, and 15-week-old seedlings were used for analysis. Needles were homogenized by pestle and mortar under liquid nitrogen, and DNA was extracted using the DNeasy w Plant Mini Kit from Qiagen AB (Sollentuna, Sweden). Changes in global DNA methylation were analyzed by the Luminometric Methylation Assay (Karimi et al., 2006), modified as described by Poborilova et al. (2015) and performed in a PyroMark Q24 instrument using Pyro Gold Reagents from Qiagen AB (Sollentuna, Sweden). In this assay, the restriction enzymes HpaII and MspI were used for methylationdependent cleavage at CCGG sites. Unmethylated CCGG sites can be cleaved by both enzymes, while neither can cleave the DNA strand if the outer C is methylated (CCGG). If the inner C is methylated (CCGG), then MspI can cleave, but not HpaII. The resulting CG-overhangs were detected by pyrosequencing analysis with pyrophosphate (PP i ) as an internal standard. The result was expressed as changes in the ratio of peak heights for (C + G) and PP i , [(C + G)/PP i ]. An increased peak height ratio corresponds to a decreased DNA methylation level. Note that the result was expressed as relative changes and not as a quantitative measure of methylation level.

Statistical analysis
Seedlings were grown in replicates and positioned in a CR design as described above. The statistical significance of the data was evaluated by analysis of variance using SPSS 20 software (SPSS Corporation). Microsoft Excel was used for computational analysis of the data. For parametric statistical tests, both Kolmogorov-Smirnov and Shapiro -Wilk tests of normality showed non-significance at the P , 5 per cent level, indicating that the distribution of data is normal. Analysis of variance (ANOVA) tests of the data were performed and the different treatment methods tested were compared using Student-Newman-Keuls (SNK) and Tukey's Honestly Significant Difference (HSD) multiple range tests at the P , 0.05 level to detect significant differences for seedling height and damaged area per attacked seedling, as well as Student's t-test for changes in DNA methylation level. For nonparametric statistical tests for number of total attacked seedlings and number of girdled seedlings, binomial tests were used to detect significant differences.

Laboratory test
Initial small-scale laboratory tests suggested a potential protecting effect of NIC treatment against pine weevil feeding on spruce seedlings grown from seeds treated with NIC and subsequently treated with NIC via watering. The results, although not statistically strong, indicated that there was somewhat less damage on treated (NIC) seedlings, when compared with control (water) seedlings. In one test, lasting for 48 h, the total area of feeding was 0.6 cm 2 on treated seedlings and 1.8 cm 2 on control seedlings. In a second test, lasting for 60 h, the corresponding values were 9.6 cm 2 (treated) and 18.1 cm 2 (control).

Field tests
Two separate field tests were carried out, one test with spruce seedlings grown from treated seeds and a second test with Seed treatment of spruce against pine weevil seedlings grown from untreated seeds, but treated via watering. Seed treatment with NIC, NIA, JA or 5-Aza did not affect seedling growth, as analyzed after 4 months, when the plants were set out in the field (Table 1; Figure 1). Neither were any effects on the vitality of the seedlings, as indicated by bud and needle status, detected.
After 2 months in the field, the extent of pine weevil gnawing on six month old spruce seedlings was observed (Tables 2 and 3). The results showed that seed treatment with NIC or JA reduced the number of seedlings attacked, while the effect of seed treatment with 5-Aza was less pronounced and seed treatment with NIA probably did not have any effect. The number of attacked seedlings was reduced by 50, 62.5 and 25 per cent by seed treatment with, respectively NIC, JA and 5-Aza (Figure 2A). Among the attacked seedlings, some were more severely damaged by girdling. The number of girdled seedlings was reduced by all treatments, 75, 100, 100 and 50 per cent reduction by seed treatment with, respectively NIC, NIA, JA and 5-Aza (Figure 2A). Although this was not statistically verifiable, the damaged area per attacked seedling showed a pattern similar to the number of attacked seedlings (Table 3), ( Figure 2B).
Treatment of seedlings grown from untreated seeds by watering with NIC or NIA did not affect plant growth as determined by the height of the seedlings before they were set out in the field (Table 4; Figure 3).
The number of attacked seedlings was reduced by 40 per cent after NIC treatment and by 53 per cent after NIA treatment, and the number of girdled seedlings were reduced by 30 and 50 per cent after treatment with, respectively, NIC and NIA, compared with control seedlings (Table 5; Figure 4A). As in the case of seed treatment, water treatment with NIC or NIA did not reduce the damaged area per attacked seedling (Table 6; Figure 4B).

DNA methylation
The effect of seed treatment with NIC on DNA methylation at a global level in needles of 15-week-old spruce seedlings grown in a greenhouse was analyzed ( Figure 5). Treatment with NIC had a reducing effect on DNA methylation, analyzed as increased cleavage of CCGG sites in DNA by the restriction enzyme MspI. This illustrates a general decrease in methylation at the outer cytosine in CCGG sites, corresponding to CXG positions in DNA. There was no difference in cleavage with HpAII between the control and treated samples, however, suggesting that the overall methylation at the inner cytosine in CCGG sites (CG positions) did not change.

Discussion
One of the most interesting aspects of stress in plants, in terms of the elicitation and priming of defensive strategies, is the question of what precise factor or condition is actually sensed by the plant, to induce a specific defensive response. Three important conditions in a plant which can be affected by stressors are oxidative stress, stability of DNA and energy state. Increased levels of reactive oxygen species arise during most types of stress, including herbivory (Kerchev et al., 2012), and often cause serious physiological damage, including strand breaks to DNA. Free NIC can be formed in response to DNA strand breaks induced by oxidative or other kinds of stress via the action of the enzyme poly(ADP-ribose) polymerase (PARP) (Schraufstätter et al., 1986;Berglund et al., 1996;Kalbin et al., 1997;Hunt et al., 2004;Surjana et al., 2010). In a negative feedback loop, NIC is also a potent inhibitor of PARP, so that NIC build-up leads to decreased NAD + cleavage (Canto et al., 2013). In addition, NIC may itself be metabolized to NAD + via the NAD + salvage pathway (Ashihara et al., 2005), a process in which the first step is metabolism of NIC to NIA by a nicotinamidase enzyme (Hunt et al., 2004). Furthermore, NIC can induce the expression of genes involved in plant defense (Berglund et al., 1993b). For instance, NIC is known to be a natural inhibitor of, but also a product of, a family of NAD + -dependent protein deacetylases, the sirtuins (Denu, 2005), which have many regulatory functions in eukaryotic cells (Canto et al., 2013). In short, an important feature of NIC in plants is that it is a key component of pathways involved in redox homeostasis as well as those involved with stress signalling and associated gene expression. Clearly, the full extent of NIC action in plant cells is highly complex, and so the precise mechanisms behind the defense-promoting properties of NIC and NIA are still unknown. Metabolism of NIC to NIA by a nicotinamidase may be an important step, and has been suggested to Figure 1 Heights of 4-month-old greenhouse-grown spruce seedlings at the time of field planting. Seedlings originated from seeds treated with water (Cont), 2.5 mM NIC, 2.5 mM NIA, 3 mM JA and 200 mM 5-Aza. Mean values based on 40 seedlings per treatment are shown. Error bars show standard deviation. Variance of treatment between groups was not significant at the P , 0.05 level. Heights of 4-month-old greenhouse-grown spruce seedlings at the time of field planting. Variance of treatment between groups was not significant at the P , 0.05 level.
Forestry function as a negative regulator of the plant hormone abscisic acid (Hunt et al., 2004). This could in turn promote defense mediated via another important plant hormone, JA (Asselbergh et al. 2008), a mechanism which is important in protection against insect attack. In the present study, NIA treatment via root uptake in spruce seedlings showed an anti-pine weevil effect in terms of the number of attacked seedlings, while there was no such effect when NIA was used for seed treatment. This differential response to NIA could simply depend on differences in NIA uptake between seeds and seedlings, but it could equally hint at different modes of action for the two compounds: it is known that NIC and NIA have similarities and differences in their biochemical effects. One main difference between the actions of these compounds is in connection with PARP and the sirtuins, discussed above. These enzymes are inhibited by NIC, but have not been shown to be inhibited by NIA (Denu, 2005). The aforementioned inhibition of sirtuins by NIC may have a strong impact on gene expression, possibly via epigenetic mechanisms, which is not induced by NIA.
Epigenetic mechanisms, such as changes in levels of DNA methylation, are closely associated with stress and defense in plants. Various types of stress induce changes in DNA methylation levels, commonly causing hypomethylation, as well as certain chromatin modifications which may serve as a 'memory' of a particular stress, improving the chance for future resistance (Sano, 2010;Jaskiewicz et al. 2011;Bräutigam et al., 2013;Kinoshita and Seki, 2014). We have previously discussed a potential role for NIC in DNA methylation processes in plant tissue, particularly the hypomethylation of DNA (Berglund, 1994;Berglund and Ohlsson, 1995;Ohlsson et al. 2013). The decreased DNA methylation levels ( Figure 5) and decreased damage by pine weevils (Figure 2A) following seed treatment with NIC, viewed alongside the similar observations of decreased pine weevil damage after seed treatment with the DNA methyltransferase inhibitor 5-Aza (Figure 2A), point at a potential involvement of general changes to DNA methylation levels in defense activation. Also supporting this connection are the results of an earlier study in which we demonstrated that UV-B exposure of indoor grown spruce seedlings caused decreased DNA methylation and increased emission of volatile terpenoids, known to influence pine weevil behaviour (Ohlsson et al. 2013). UV-B exposure has also been shown to increase the level of both NIC and trigonelline (N-methyl nicotinic acid) in plant tissue (Berglund et al. 1996). Trigonelline is formed from NIA, which in turn is formed from NIC by the action of nicotinamidase. It has been shown that trigonelline can promote anti-microbial defense in plants in association with a decrease in global DNA methylation (Kraska and Schönbeck 1993). In line with this, we consider it a possibility that the hypomethylation of DNA in plants grown from seeds treated with NIC could also serve to promote defense against herbivorous insects, resulting specifically in this case in a reduction in pine weevil attacks. In future studies, we would also like to include marker gene expression analysis to investigate a possible connection between DNA methylation and spruce defence induction.
Many reports point at the importance of the mother plant for resistance and adaptive responses in the next generation (Holeski et al., 2012;Pastor et al. 2013), transmitted via effects on the embryo or seed (Yakovlev et al., 2011;Worrall et al., 2012;Bräutigam et al., 2013). It is possible that exogenous application of native signalling compounds (or close synthetic mimics thereof) directly to the seed may mimic such information transfer from the mother plant to the embryo/seed, providing the young plant with the capacity to adapt to stressful changes in the environment. Although the nature of this signalling system is not yet well understood, epigenetic mechanisms are likely involved (Yakovlev et al., 2011;Bräutigam et al., 2013). Seed treatment over a matter of a few hours with for example NIC can influence the properties of the plant several months later, rather than inducing only transient physiological changes as might be expected. A plausible explanation is that physical changes to the plant's DNA have been made, thereby altering the epigenetic coding capacity of the organism, and that this information is therefore carried through many cell divisions. Differences between nonattacked and attacked seedlings, and between nongirdled and girdled seedlings were significant at the P , 0.05 level. Damaged area per attacked seedling by pine weevils on 6-month-old spruce seedlings. Variance of treatment between groups was not significant at the P , 0.05 level.
Seed treatment of spruce against pine weevil A major focus of research regarding induced defense against pine weevils in conifers has until now been concentrated on the effects of jasmonates sprayed onto plants (Holopainen et al., 2009;Zas et al., 2014). A recent field study showed that MeJA spraying of seedlings can promote defense against pine weevil attack in pine, and to a lesser extent also in spruce . A major drawback of MeJA treatment via spraying appears to be a decreased plant growth, including decreased secondary growth, in conifer seedlings (Moreira et al., 2012). However, watering of conifer seedlings with MeJA has also been studied and resulted in increased terpenoid levels, mainly in roots and stems (Huber et al., 2005). An increased resin formation in stems has been shown to follow MeJA treatment, and stems or stem pieces from such plants presented to pine weevils are less extensively gnawed than stem pieces from untreated plants (Holopainen et al., 2009;Moreira et al., 2012;Zas et al., 2014). However, an increased resin content of MeJA treated trees is considered a drawback regarding wood quality (Holopainen et al., 2009). Interestingly, a study by Moreira et al. (2014) shows that in several pine species there is a trade-off between constitutive and inducible tissue non-volatile resin content, depending on geographical and climatic factors. With the seed treatment approach presented in this report, we intended to develop a simple and mild treatment method that would have a minimal effect on growth. The indication that JA may be a defense-potentiating compound in spruce via seed treatment has to be investigated further in association with its metabolite, the long distance signal MeJA.
This study shows that seed treatment with the natural, nontoxic, and within this context novel, compounds NIC and NIA (also known as niacin or vitamin B 3 ), in addition to the better known defense-inducing compound JA, can promote defense against pine weevil attack in spruce. For practical use of the  Heights of 1.5-year-old spruce seedlings at the time of field planting, after treatment via watering for 2.5 months. Variance of treatment between groups was not significant at the P , 0.05 level.

Figure 3
Heights of 1.5-year-old spruce seedlings at the time of field planting, after treatment via watering for 2.5 months. Seedlings were treated with water (Cont), 2 mM NIC or 2 mM NIA. Mean values based on 30 seedlings per treatment are shown. Error bars show standard deviation. Variance of treatment between the groups was not significant at the P , 0.05 level.
compounds for spruce protection, toxicological evaluations have to be performed. Seed or seedling treatment with natural substances is an attractive alternative to the comprehensive use of toxic synthetic pesticides and genetically modified plants, although these strategies remain important. It may be that the extent of pine weevil damage to seedlings depends on the attraction the insects feel towards the plants, as well as nutritional suitability of the stem tissue for feeding. We interpret the frequency of attacked plants as a measure of pine weevil attraction by volatile compounds, and the extent of damage as a measure of the tastiness of the stem cambium/ phloem for the weevils. The decreased number of attacked seedlings seen in the present study could be due to a lower attractiveness for pine weevils. The extent of girdling, which is detrimental to the seedlings, was lower in seedlings from treated seeds compared with the control in the present study. A low frequency of girdling may reflect that the insect, instead of staying at one place for continuous feeding, searches for a more tasty/attractive part of the stem. This behaviour would decrease the risk of girdling, even if the total area of damage may be considerable. This first trial regarding protection of spruce seedlings against pine weevil attack via seed treatment or watering only covers the first season in the field, a time which is decisive for the attractiveness of the plants to the Differences between nonattacked and attacked seedlings, and between nongirdled and girdled seedlings were significant at the P , 0.05 level.

Figure 4
Attack by pine weevils on 1.5-year-old spruce seedlings in a field test after treatment of seedlings via watering for 2.5 months. Seedlings were treated with water (Cont), 2 mM NIC or 2 mM NIA. Fifty-five seedlings per treatment were examined. (A) Number of attacked and girdled seedlings. (B) Damaged area per attacked seedling; variance of treatment between the groups was not significant at the P , 0.05 level for any condition. Damaged area per attacked seedling by pine weevils on 1.5-year-old spruce seedlings. Variance of treatment between groups was not significant at the P , 0.05 level.
Seed treatment of spruce against pine weevil insects, and thereby for the survival of the plants. Further optimization of the strategies outlined here, regarding the duration of seed treatment, the time period between treatment and sowing, the concentration of compounds used, and other factors, will hopefully lead to more improvements in seedling protection. Therefore, the results of the present investigation should not be seen as a ready to use concept for conifer protection against pine weevils, but rather the opening of a door into new defensive strategies.

Conclusion
In the present study, we point at a new strategy for future research aiming at improved forest protection and environmentally friendly forestry. This investigation indicates that seed treatment and watering of young spruce seedlings with selected nontoxic plant compounds, especially NIC, can give protection against attack by pine weevils in the field. The results could point at a role for epigenetic mechanisms in this process. The results also support a potential importance of NIC and NIA as defense signal mediating compounds as originally suggested by Berglund (1994).

Figure 5
Effect of NIC (2.5 mM) seed treatment on DNA methylation changes in needles of 15-week-old spruce seedlings. DNA was cleaved by the restriction enzymes MspI and HpaII and analyzed by LUMA. A higher value for [(C + G)/PP i ] ratio corresponds to a lower degree of DNA methylation. An asterisk (*) corresponds to a significant difference from the control at the P , 0.05 level, as determined by an unpaired t test.