Thomas W. Kimmerer

Nutritional quality of specific leaf tissues and selective feeding by a specialist leafminer

SummaryMany folivorous insects are selective feeders which consume specific leaf tissues. For specialist herbivores feeding on plants of overall low nutritional quality, selective feeding may allow consumption of a high quality resource. Selective feeding may also allow insects to avoid structural or allelochemical defenses. We examined the structure and chemistry of leaves of American holly, Ilexopaca Aiton, and the feeding site of its principal insect herbivore, the native holly leafminer, Phytomyza ilicicola Loew (Diptera: Agromyzidae), to test the hypothesis that the leafminer consumes tissues which are of greater nutritional quality than the leaf as a whole. Holly leaves have a continuous layer of palisade mesophyll, uninterrupted by fibers or vascular bundles. The leafminer feeds entirely within this layer. The palisade mesophyll contained 196 mg/g dry wt extractable protein, more than twice as much as the leaf as a whole, and 375 mg/g dry wt saponins, more than 9 times that of the leaf as a whole. The water content of the palisade mesophyll was 66% higher than that of the leaf as a whole. The palisade mesophyll is 3–4 cell layers thick in leaves grown in full sun, but only 2 layers thick in shaded leaves. Crystals, probably of calcium oxalate, are abundant in the abaxial cell layer. These may impose mechanical constraints on larval feeding in shade leaves, which are thinner than sun leaves. Selective feeding on the middle palisade mesophyll of sun leaves allows the leafminer to consume a resource which is lacking in mechanical barriers and is rich in protein and water, but which contains large amounts of saponins.

Metabolism of Transpired Ethanol by Eastern Cottonwood (Populus deltoides Bartr.)

Ethanol has previously been shown to be present in the xylem sap of flooded and nonflooded trees. Because of the constitutive presence of alcohol dehydrogenase in the mature leaves of woody plants, we hypothesized that the leaves and shoots of trees had the ability to metabolize ethanol supplied by the transpiration stream. 1-[14C]Ethanol was supplied to excised leaves and shoots of eastern cottonwood (Populus deltoides Bartr.) in short- and long-term experiments. More than 99% of the radiolabel was incorporated into plant tissue in short-term experiments, with more than 95% of the label remaining in plant tissue after 24 h. In all experiments, less than 5% of the label was transpired as ethanol and less than 1% was emitted as CO2. In excised leaf experiments, less than 0.5% of the radiolabel escaped from the leaf. Fifty percent of the label was incorporated into the petioles of excised leaves; 56% was incorporated into the stems of excised shoots. Very little label reached the leaf mesophyll cells of excised shoots, as revealed by autoradiography. Radiolabel appeared primarily in the water- and chloroform-soluble fractions in short-term experiments, whereas in long-term experiments, label was also incorporated into protein. These results demonstrate that the leaves and stems of trees appear to have substantial ability to scavenge ethanol from the transpiration stream, allowing efficient recovery of ethanol produced elsewhere by hypoxic tissues. When labeled ethanol was supplied to excised petioles in a 5-min pulse, 41% of the label was incorporated into organic acids. Some label was also incorporated into amino acids, protein, and the chloroform-soluble fraction, with very little appearing in neutral sugars, starch, or the insoluble pellet. Labeled organic acids were separated by high performance liquid chromatography and were composed of acetate, isocitrate, [alpha]-ketoglutarate, and succinate. There was no apparent incorporation of label into phosphorylated compounds. We conclude that, in higher plants, ethanol is metabolized to acetaldehyde and then to acetate by alcohol and aldehyde dehydrogenases, and then into general metabolism.

Carbohydrate reserves, radial growth, and mechanisms of resistance of oak trees to phloem-boring insects.

SummaryThe twolined chestnut borer, Agrilus bilineatus (Weber) (Coleoptera: Buprestidae), attacks oaks (Quercus spp.) that have been weakened by prior environmental or biotic stress. Our earlier work showed that trees with relatively low winter starch reserves are more likely to be attacked by A. bilineatus the following summer. We hypothesized that such trees may have less energy available for defense (Callus formation and allelo-chemical synthesis) in tissues wounded by borer larvae. However, wounding experiments showed little or no relationship between winter or summer carbohydrate reserves, callus formation, radial growth, or concentrations of tannins and phenolics in wounded or nonwounded phloem tissues. Trees with relatively low winter carbohydrate reserves were again found to be attractive to adult A. bilineatus, although not all low starch trees were attacked or successfully colonized by borers. There was a trend for carpenterworm larvae, Prinoxystus robiniae (Lepidoptera: Cossidae), a generalist bark and wood borer, to be more successful in establishing galleries on low starch trees. Carpenterworms gained significantly more weight when fed phloem from trees attractive to A. bilineatus. Oaks that attracted large numbers of A. bilineatus or that were successfully colonized by the borer produced significantly less callus than did non-attacked trees when experimentally wounded at about the time of Agrilus egg hatch. Callus formation may limit the establishment of small larvae that feed slowly in the cambial region. These results indicate that current theory regarding relationships between increased tree stress and decreased allocation of energy reserves to radial growth and defense against phloem borers may be an oversimplification. We suggest that tree growth and the defensive response of phloem tissues may be limited more by the rate of carbohydrate utilization or by changes in source-sink relationships than by storage levels. Callus formation and synthesis of allelochemicals in wounded phloem may be under the same control as cambial activation, which is mediated by plant growth regulators and can be influenced by environmental conditions.

Intraspecific variation in production of astringent phenolics over a vegetation-resource availability gradient

SummaryThe chemical constituency of flowering dogwood (Cornus florida L.) and red maple (Acer rubrum L.) foliage was analyzed over a species compositional gradient to test the hypothesis that over subtle gradients of moisture and nutrient availability production of phenolic compounds will be increased on sites of greatest stress. Calcium and nitrogen concentrations declined along the gradient in both species, while phosphorus showed a significant decline only in red maple. Lignin concentrations in both species were unrelated to the vegetation gradient, but astringent phenolics increased by 156% and 159% in dogwood and red maple, respectively. The correlation between production of polyphenolds and site quality supports previous observations that under conditions of environmental stress production of many secondary compounds is increased, and suggests that this relationship is significant over subtle environmental gradients.

Inhibition of herbivory on young holly leaves: evidence for the defensive role of saponins

SummaryResponse of the southern red mite, Oligonychus ilicis McGregor, to young and one year old leaves of Ilex opaca Aiton was studied on three dates during the period of leaf expansion in the spring. Young foliage, which is rich in nutrients but also contains high levels of saponins, was found to be unsuitable for colonization by this oligophagous herbivore until the leaves had matured and levels of saponins had declined. Mites preferentially colonized and had higher survival and reproduction on sclerophyllous, one-year old leaves than on young leaves in early spring. High levels of saponins in young, second-flush leaves of shoots that had earlier been damaged by frost were again correlated with low mite survival in June. Laboratory preference tests with the fall webworm, Hyphantria cunea Drury, and the eastern tent caterpillar, Malacosoma americanum (F.), indicated that young, saponin-rich holly foliage is especially unpalatable to these highly polyphagous caterpillars. Fifth instar fall webworms required significantly longer to complete their development, attained lower weights, and had greater mortality on artificial diet amended with low concentrations of purified holly saponins than on control diet. These results support the hypothesis that the high levels of saponins in young holly leaves provide protection from herbovores until the leaves have matured and their structural defenses are developed.

The role of host tree condition in attack of white oaks by the twolined chestnut borer, Agrilus bilineatus (Weber) (Coleoptera: Buprestidae)

SummaryThe twolined chestnut borer, Agrilus bilineatus (Weber) (Coleoptera: Buprestidae), attacks stressed oaks (Quercus spp.) and is associated with extensive mortality of trees in the eastern deciduous forests of North America. We examined host location by the insect and subsequent host mortality in experimentally stressed trees. A. bilineatus adults were able to rapidly and specifically locate stressed oak trees. Up to 160 beetles per week were captured on sticky band traps on the trunks of stressed trees, while beetles rarely landed on unstressed control trees. This suggests that adult borers have an acute perception of host tree “quality”, and that this perception is from a distance. One mechanism of host location may be detection of volatile compounds produced by stressed trees.The condition of the host tree appears to regulate both beetle attraction and successful colonization. Mortally wounded (xylem-girdled) trees attracted beetles only until the cambium died. Xylem-girdled trees were attacked early in the beetle flight season, but larvae did not survive to emerge as adults from these trees. In contrast, phloemgirdled trees continued to attract beetles throughout the flight period. Phloem-girdled trees which were heavily attacked by A. bilineatus died late in the season in which they were attacked. Lightly attacked trees survived until the following growing season, and were then heavily attacked and killed. In one stand, phloem-girdled trees were not attacked, healed over the girdling wounds and were still alive three years after girdling. These results indicate that oak trees are only attractive to A. bilineatus within a narrow range of physiological conditions following stress but prior to mortality. A. bilineatus appears to be a proximate agent of mortality in stressed oaks in eastern North America.

Seasonal allocation of defense investment in Ilex opaca Aiton and constraints on a specialist leafminer

SummaryPhytomyza ilicicola (Diptera: Agromyzidae), a univoltine specialist leafminer, is one of the few insect herbivores of American holly. Adult emergence is closely synchronized with leaf flush in spring, and females make numerous feeding punctures on and oviposit in new leaves. Larvae hatch in late May and June, but their feeding period and development are prolonged so that more than 80% of the mine enlargement occurs from January until March of the following year. We propose that this unusual life cycle reflects adaptation to constraints imposed by seasonal and age-related changes in chemical and structural defenses, and in nutritional quality of holly foliage. As holly leaves age, there is a shift in allocation of defense investment away from allelochemicals, including phenolic compounds and saponins, toward leaf sclerophylly, spinose teeth, and low foliar nitrogen and water. Rapid increases in leaf toughness and decreases in nutritional quality limit availability of leaf tissues for adult feeding and oviposition to a two-to threeweek phenological window during leaf flush. Mature holly foliage is a nutritionally poor resource by nearly all criteria known to affect food quality for herbivores. This may be the main reason for the prolonged larval development of P. ilicicola. Alternatively, winter feeding and pupation in spring may be adaptations which help to ensure synchrony of adult emergence with leaf flush. Repeated puncturing by female P. ilicicola does not render leaves more suitable for larvae, nor is it a means by which females sample leaf exudate to assess leaf quality prior to oviposition. Rather, leaf puncturing occurs mostly on leaves that are relatively high in soluble nitrogen, and is apparently a means by which females obtain protein and sugars prior to and during oviposition.

Do holly leaf spines really deter herbivory

SummaryAlthough spinose teeth of holly leaves have been widely cited as an example of a physical defense against herbivores, this assumption is based largely on circumstantial evidence and on general misinterpretation of a single, earlier experiment. We studied the response of third and fifth instar larvae of the fall webworm, Hyphantria cunea Drury, a generalist, edge-feeding caterpillar, to intact American holly leaves and to leaves that had been modified by blunting the spines, by removing sections of leaf margin between the spines, or by removing the entire leaf margin. The results suggest that the thick glabrous cuticle and tough leaf margin of Ilex opaca are more important than the spinose teeth in deterring edge-feeding caterpillars. Microscopic examination of mature leaves revealed that the epidermis is thickened at the leaf margin, and that the leaf is cirucumscribed by a pair of fibrous veins. In simple choice tests neither domesticated rabbits nor captive whitetailed deer discriminated between spinescent holly foliage and foliage from which spines were removed. Nevertheless, we found little evidence of herbivory by mammals in the field, either on small experimental trees or in the forest understory. While it is possible that spinose teeth contribute to defense by reducing acceptibility of holly relative to other palatable plant species, we suggest that the high concentrations of saponins and poor nutritional quality of holly foliage may be more important than spines in deterring vertebrate herbivores. The degree of leaf spinescence and herbivory was compared at different heights with the tree canopy to test the prediction that lower leaves should be more spinescent as a deterrent to browsers. Leaves on lower branches of mature forest trees were slightly more spinescent than were upper leaves, and juvenile trees were slightly more spinescent than were mature trees. However, there was no relationship between degree of spinescence and feeding damage. The greater spinescence of holly leaves low in the canopy is probably an ontogenetic phenomenon rather than a facultative defense against browsers.

Molecular genetics and physiology of alcohol dehydrogenase in woody plants.

Abstract Alcohol dehydrogenase (ADH) is an enzyme that plays a central role in the anaerobic metabolism of plants. In herbaceous and woody plants, ADH activity occurs in root apices and in germinating seeds. In trees, ADH activity also occurs in leaves and in stems, and it is particularly high in the vascular cambium, a site of ethanol synthesis. We are investigating the biochemical properties and physiological functions of ADH in trees, as well as the environmental and developmental circumstances in which ADH is expressed. ADH genes from a number of herbaceous angiosperms have been isolated and characterized, and specific DNA sequences have been implicated in regulating ADH gene expression. This information provides a good foundation from which we are beginning to characterize the regulation of ADH genes in hardwoods and conifers. The ability to manipulate ADH genes using molecular technology will facilitate detailed explorations of the unusual physiology and biochemistry of ADH expression in woody plants. Such experiments are especially important in trees because of the apparently widespread occurrence of hypoxic conditions in their tissues.

Winter starch reserves of white oak as a predictor of attack by the twolined chestnut borer, Agrilus bilineatus (Weber) (Coleoptera: Buprestidae)

SummaryThe twolined chestnut borer, Agrilus bilineatus (Weber) (Coleoptera: Buprestidae), attacks oaks (Quercus spp.) and is associated with extensive mortality of trees in the eastern deciduous forests of North America. We tested the hypothesis that winter starch reserves of oak roots are an indicator of tree vigor and that only trees low in stored starch would be attacked by A. bilineatus. We measured the levels of stored starch in the roots of 200 non-infested healthy white oaks during the dormant season and determined their correlation with A. bilineatus attacks the following spring. There was a significant increase in A. bilineatus captures on sticky traps with a decrease in winter starch reserves. Trees low in stored starch that were also stressed by phloem-girdling attracted 3.7 times as many beetles as did non-girdled trees that were low in starch. However, non-girdled trees that had low winter starch reserves were also attacked. Only oaks that had had extremely low winter root starch reserves (<5mg/g dry weight of root sapwood tissue) were heavily attacked by A. bilineatus and subsequently died. One third of non-girdled low starch trees and 67% of phloem-girdled low starch trees died, whereas none of the trees with root starch >5 mg/g dry wt died. These results indicate that winter starch reserves are a good predictor of A. bilineatus attack.