Riitta Julkunen-Tiitto

HERBIVORE RESISTANCE IN BETULA PENDULA: EFFECT OF FERTILIZATION, DEFOLIATION, AND PLANT GENOTYPE

Plant resistance to herbivores is affected both by genetic and environmental factors. The carbon–nutrient balance hypothesis (CNB) explains environmentally induced variation in both constitutive and delayed herbivore-induced resistance (DIR) in terms of variation in soil fertility and light regime. The CNB hypothesis predicts that an increase in the availability of nutrients (e.g., fertilization) decreases both constitutive and induced resistance against herbivores. We tested the relative roles of plant genotype, defoliation, and soil fertility in determining herbivore resistance of cloned silver birch Betula pendula Roth saplings. As indicators of insect and mammalian resistance we conducted bioassays with a geometrid moth, Epirrita autumnata (Borkhausen), and counted the resin droplets on the shoot of the saplings, respectively. In addition, we measured rapid induced resistance (RIR) against the insect herbivore. Finally, we analyzed leaf secondary chemistry to investigate the correlations of secondary chemicals with the level of resistance measured using the performance of E. autumnata. With respect to the constitutive resistance against an insect herbivore, our results support the CNB hypothesis; the larvae of E. autumnata had a higher relative growth rate and pupal mass on fertilized saplings compared to nonfertilized saplings, i.e., the fertilized saplings had a lower resistance level. However, the relative growth rate of E. autumnata was significantly decreased by defoliation only when the larvae were grown on fertilized saplings. The number of resin droplets increased due to fertilization and, in fertilized saplings, following defoliation, but these responses were highly determined by the genotype of the sapling. Altogether, the results on resin droplets are not in accordance with the CNB hypothesis. The concentration of condensed tannins correlated negatively with E. autumnata growth rate and pupal mass in both fertilization levels, whereas the concentration of total nontannin phenolics correlated positively with the E. autumnata growth rate in nonfertilized saplings. In addition, the concentration of myricetin glycosides correlated negatively with the pupal mass of E. autumnata, whereas the correlations between E. autumnata performance indices and other groups of flavonol glycosides were either significantly positive (kaempferol glycosides) or nonsignificant (quercetin glycosides). Further, the concentration of 3,4′-dihydroxypropiophenone 3-glucoside (DHPPG) correlated positively with the magnitude of induction in E. autumnata growth rate and pupal mass in fertilized saplings, where the significant induction in resistance occurred. The correlations of secondary chemistry and E. autumnata performance indices suggest that the constitutive level of resistance of B. pendula against E. autumnata is mainly determined by the concentration of condensed tannins, whereas the induced resistance is determined by the concentration of nontannin phenolics, such as flavonol glycosides and DHPPG. We observed significant differences among the clones in their insect and mammalian resistance (i.e., genetic basis for the resistance), which indicates that resistance can evolve as a response to herbivory. However, fertilization explained a higher proportion of variance in insect performance indices than the genotype of the plant, whereas the opposite was true for the amount of resin droplets, which we used as an indicator of mammalian resistance.

Testing the effects of drying methods on willow flavonoids, tannins, and salicylates.

In this study, we compared the effects of several preservation methods on the secondary phenolics of the mature leaves of purple willow (Salix purpurea L., Salicaceae) with results obtained with fresh leaf analyses. Conventional freeze-drying, in which the leaves were first frozen with liquid nitrogen and then placed in a freeze-dryer, produced substantial qualitative and quantitative changes in purple willow flavonoids and salicylates. Modified freeze-drying, in which leaves were put into a freeze-dryer without being prefrozen, gave concentrations that, for most secondary components, were comparable with those found in fresh leaves. Reducing the freeze-dryer chamber temperature hindered the decomposition of phenolics in prefrozen leaves and in leaves dried without prefreezing. Heat drying induced substantial changes in the composition of all phenolics, except for apigenin-7-glucoside. Vacuum drying at room temperature gave the highest concentrations for nearly all phenolics, while room-drying with desiccation gave results that were comparable with those obtained by fresh leaf analyses.

Trade-offs in phenolic metabolism of silver birch: Effects of fertilization, defoliation, and genotype

We examined the chemical responses of 10 silver birch (Betula pendula) clones to fertilization and defoliation in a field experiment. In defoliation, every second leaf was removed from the saplings. Three days later, two undamaged short-shoot leaves were collected, air-dried, and analyzed for condensed tannins and 34 nontannin phenolic compounds by high-performance liquid chromatography. The clones showed substantial variation in phenolic composition of the leaves and in chemical responses to fertilization and defoliation. A cluster analysis by UPGMA indicated that the phenolic profiles of birch leaves were affected more by genotype than fertilization or defoliation, and the clones could thus be distinguished from each other. In addition, on the basis of their overall phenolic composition, the clones were clustered loosely in three clone groups. The leaves of fertilized saplings contained lower levels of condensed tannins than controls, as predicted by carbon/nutrient balance (CNB) hypothesis. However, fertilization had no effect on the total amount of nontannin phenolics. The concentrations of (+)-catechin, 3,4′-dihydroxypropiophenone 3-glucoside (DHPPG), 3-cinnamoylquinic acids, and flavone aglycones were lower in fertilized saplings, whereas the opposite was true for 5-cinnamoylquinic acids and the total amount of flavonol glycosides. Although our results provide support for the CNB hypothesis, they also show that the accumulation of phenolic compounds in birch leaves is strongly coordinated. Different branches of the biosynthetic pathway of phenolic compounds may compete for substrates, and such internal metabolic trade-offs may explain the differential accumulation of the compounds. In fertilized saplings, the concentration of condensed tannins was also negatively correlated with the amount of triterpenoid resin droplets measured from the same saplings. We suggest that a linkage via malonyl-CoA between the biosynthetic routes to terpenoids and flavonoid derivatives, such as condensed tannins, may explain the different responses to fertilization reported for terpenoids and phenolics. Undamaged leaves of partially defoliated saplings contained more DHPPG and flavone aglycones and less cinnamic acid derivatives and (+)-catechin than did leaves of control saplings. The induction of DHPPG and flavonoid aglycones was significantly and negatively correlated with the concentration of myricetin glycosides in fertilized saplings, which may indicate a trade-off between induced and constitutive defense. Moreover, in fertilized saplings, the three clone groups formed by UPGMA clustering differed significantly in the magnitude of induction of DHPPG and flavone aglycones. Different birch genotypes may thus have different modes of chemical defense, and the magnitude of chemical response of a genotype may partly depend on resource availability. In general, our results show that new insights in the theory of chemical defense can be gained by accomplishing studies on plant–herbivore interaction with high chemical resolution.

Chemical diversity of several Betulaceae species: comparison of phenolics and terpenoids in northern birch stems

Abstract Phenolics and terpenoids characteristic for silver birch (Betula pendula Roth) were screened in current-growth twigs of seedlings (1-growing-season-old individuals) and current-growth twigs of saplings (3 to 5-year-old individuals) of several birch species. Seedlings were grown in a greenhouse for 1 growing season. Saplings were field cultivated or wild-stand individuals. Chemicals were methanol-extracted, purified and HPLC-diode array detector-, gas chromatograph-flame ionizaton detector- and gas chromatograph-mass selective-analyzed. Species-specific qualitative and quantitative variation of secondary chemicals in birch stems were considerable and dependent on the age of the individual. The chemical diversity and amount were lower in seedlings than in saplings. In saplings, (+)-catechin and its derivatives were found in nearly all species, while arylbutanoids (±rhododendrol glucosides), an arylheptanoid (platyphylloside) and dammaraneterpenoids (papyriferic acid, deacetylpapyriferic acid and pendulic acid) showed a more restricted distribution. The chemicals analyzed in birch stems are a useful tool for recognition of species.

Ontogenic development of chemical defense by seedling resin birch : energy cost of defense production

Whether production of chemical defenses by plants is or is not an energetically costly process is an important, but unresolved, question in chemical ecology. We suggest studies of the ontogenetic development of plant defense systems can help resolve the question. As an example of this approach to the cost question, we explore the problems associated with production of immobile chemical defenses that defend juvenile resin birches against browsing by mammals. From this exploration we draw two conclusions: (1) Shortly after germination, production of chemical defenses by small-seeded species, such as birch, is energetically costly. (2) Opposing selection for defense versus competitive ability in the seedling stage of birch has resulted in a trade-off in allocation of carbon to production of immobile chemical defense versus allocation of carbon to production of storage reserves. We suggest this trade-off results in a large indirect cost of defense because carbon used for production of immobile chemical defenses is unavailable for support of growth in the future, but stored carbon can be used to support future growth.

A chemotaxonomic survey of phenolics in leaves of northern salicaceae species

Abstract The leaves of 15 Salicaceae species were screened for total phenolics, condensed tannins, catechins and phenolic glycosides. The ability to produce phenolics varies widely between willow species. All species contain condensed tannins and phenolic glycosides, although some species showed only trace amounts. Five of the 15 species lack catechins. It was possible to distinguish each willow species on the basis of its leaf phenolic glycosides.

Phenological variation as protection against defoliating insects: the case of Quercus robur and Operophtera brumata.

Phenological synchrony between budburst and emergence of larvae is critical for the fitness of many spring-feeding insect herbivores. Therefore, large intraspecific variation in timing of budburst of the host may have a negative effect on the herbivore. We studied how asynchrony between emergence of larvae and budburst affects the fitness of Operophtera brumata (Lepidoptera: Geometridae), a major defoliator of Quercus robur, which can adapt to the phenology of a single tree. It is known that, in maturing leaves of Q. robur, accumulation of condensed tannins has a negative effect on growth of O. brumata. However, there is no information about the effect of hydrolysable tannins and other phenolics that are potential antifeedants. In this study, we also analysed changes in secondary chemistry of the foliage of Q. robur and how different compounds are correlated with growth and survival of O. brumata. The effect of asynchrony on O. brumata was studied in rearing experiments. The neonate larvae were incubated without food for different periods of time. The decline in nutritional quality of foliage was estimated by rearing cohorts of larvae with manipulated hatching times on the leaves of ten individual Q. robur trees. For the chemical analysis, the foliage of these trees was sampled at regular intervals. In the absence of foliage, mortality of neonate larvae started to increase exponentially soon after the larvae emerged. If the larvae missed budburst, the decline in nutritional quality of the foliage led to increased mortality and lower body mass (= fecundity). Hydrolysable tannins were not significantly correlated with performance of the larvae. Only condensed tannins were found to correlate negatively with the growth and survival of O. brumata. Certain individual trees were unsuitable hosts for O. brumata because the decline in quality of the foliage was very rapid. Based on regression equations for increasing rate of mortality and decreasing fecundity, we calculated that a relatively small mismatch of ±30 degree days between budburst and hatching of larvae leads to a 50% decrease in the fitness of O. brumata. Thus, large phenological variation within a Q. robur stand can limit the colonisation of neighbouring trees by dispersing larvae. Furthermore, the hybridisation of moths adapted to phenologically different trees may lead to maladapted phenology of their offspring.

Variation in Phenolic Compounds within a Birch (Betula pendula) Population

In previous studies, the qualitative and quantitative variation found in defense chemistry among birch populations and even among individual clones has been considerable. However, information about variation among adult, naturally regenerated birch trees from natural populations is still lacking. In this study, the phenolic composition of leaves of 30 naturally regenerated 20-year-old birch (Betula pendula) trees was analyzed for two successive years in order to characterize the chemical composition of individual trees, analyze the annual variation, and determine chemical similarities among individual trees within a population. The main phenolic compounds were flavonoid glycosides, myricetin, and quercetin derivatives. Annual variation in concentration among leaves was large. In most trees, concentrations were markedly higher in 1998 than in 1997; for certain compounds, the detected increase was as much as a 50%. However, for some individual trees, there were no differences between years in chemical quantity. Thus, when selection or grouping of trees is based on secondary chemistry, quantitative variation should be considered carefully. With the qualitative UPGMA method of classification, four chemotypes were found. The grouping was similar for both years, and qualitatively the results of an individual tree seem to be independent of sampling year. The stability in chemical profile of individual trees suggests that quality is tightly controlled by genotype, which provides a recognition tool for chemotaxonomy. The high within-population variation found in leaf defense chemistry may provide protection against different types of insects (generalists or specialists) and, thus, have positive effects on population survival.

High-performance liquid chromatographic determination of flavonoids in Betula pendula and Betula pubescens leaves

Abstract A high-performance liquid chromatography (HPLC) procedure based on a water–methanol gradient with tetrahydrofuran for simultaneous analysis of flavonoids and other phenolics of differing polarities in birch leaves was developed. Mobile phases with and without tetrahydrofuran or orthophosphoric acid provided additional information for tentative identification of the compounds. Forty-five compounds were determined from Betula pendula and B. pubescens leaves. Five of the isolated flavonol glycosides have not been reported earlier from B. pendula. B. pendula leaves have a range of myricetin and quercetin glycosides with the same sugar moieties in roughly the same proportions. Flavonoid aglycones were deposited on leaf surfaces.

HOST PREFERENCE AND LARVAL PERFORMANCE OF THE SALICYLATE‐USING LEAF BEETLE PHRATORA VITELLINAE

Larvae of Phratora vitellinae (Coleoptera: Chrysomelidae) convert salicyl glucosides from the host plant into a larval defensive secretion with salicylaldehyde. This secretion repels generalist predators. Willows vary greatly in the concentrations of salicyl glucosides in their leaves. One may predict that P. vitellinae prefers and survives better on plants that contain more salicyl glucosides. We determined the amount of larval secretion, host preference, larval growth, and larval survival of P. vitellinae on Salix myrsinifolia, S. pentandra, and S. phylicifolia. We also measured feeding rates of three natural predators on P. vitellinae larvae feeding on different hosts. Salix pentandra and S. myrsinifolia contained substantial amounts of salicyl glucosides, but S. phylicifolia contained very little of them. Phratora vitellinae larvae produced more secretion on S. pentandra than on S. myrsinifolia. They produced little secretion on S. phylicifolia. Adult beetles preferred S. myrsinifolia over S. pentandra and S. pentandra over S. phylicifolia. Larvae grew most rapidly on S. myrsinifolia and S. pentandra. Their growth was slowest on S. phylicifolia. The larval survival was similar on S. myrsinifolia and S. phylicifolia, but it was significantly lower on S. pentandra. The natural predators fed equally well on P. vitellinae feeding on S. myrsinifolia and S. phylicifolia. Thus, the host preference of P. vitellinae did not cor- respond to larval survival on these hosts, but rather to larval growth. Larval survival of P. vitellinae was not related to the amount of defensive secretion. Natural predators were not repelled by the host-derived defensive secretion. We discuss the implications of these findings for the evolution of host plant use in this herbivore.