Defoliation-induced responses in peroxidases,phenolics, and polyamines in scots pine (Pinus sylvestris L.) needles

Effects of artificial defoliation on defensive needle chemistry in Scots pine (Pinus sylvestris L.) were evaluated with particular emphasis on peroxidases, phenolic compounds, soluble sugars, polyamines, and foliar nitrogen levels. The study was carried out on a nutrient-poor Scots pine stand with 8- to 25-year-old trees. Defoliation treatment consisted of repeated defoliation in two successive years with respective control trees. Defoliation was done before needle flushing by removing all mature needles. Guaiacol peroxidase activity increased in the needles after the first defoliation. The difference between treatments diminished towards autumn, and disappeared before the second defoliation in the next summer. After the second defoliation, the activities showed a similar trend. Apparently, peroxidases are involved in inducible chemical changes and recovery reactions that occur in the intact needles shortly after defoliation. After the second defoliation, total nitrogen concentration in the current year needles was about 20% lower, and free putrescine (a polyamine) concentration was 40% lower in the defoliated trees than in control needles. These changes indicate a loss of nitrogen due to defoliation. Specific phenolic compounds such as quercitrin, (+)-catechin, and two catechin derivatives increased in current year needles in response to defoliation. Accumulation of starch and sucrose in the current year needles of repeatedly defoliated trees may imply decreased assimilate transport. The results are indicative that changes in needle phytochemistry in response to defoliation accompany changes in needle nitrogen metabolism.     

Effects of biotic and abiotic stress on induced accumulation of terpenes and phenolics in red pines inoculated with bark beetle-vectored fungus

This study characterized the chemical response of healthy red pine to artificial inoculation with the bark beetle-vectored fungusLeptographium terebrantis. In addition, we sought to determine whether stress altered this induced response and to understand the implications of these interactions to the study of decline diseases. Twenty-five-year-old trees responded to mechanical wounding or inoculation withL. terebrantis by producing resinous reaction lesions in the phloem. Aseptically wounded and wound-inoculated phloem contained higher concentrations of phenolics than did constitutive tissue. Trees inoculated withL. terebrantis also contained higher concentrations of six monoterpenes,-pinene,-pinene, 3-carene, limonene, camphene, and myrcene, and higher total monoterpenes than did trees that were mechanically wounded or left unwounded. Concentrations of these monoterpenes increased with time after inoculation. Total phenolic concentrations in unwounded stem tissue did not differ between healthy and root-diseased trees. Likewise, constitutive monoterpene concentrations in stem phloem were similar between healthy and root-diseased trees. However, when stem phloem tissue was challenged with fungal inoculations, reaction tissue from root-diseased trees contained lower concentrations of-pinene, the predominant monoterpene in red pine, than did reaction tissue from healthy trees. Seedlings stressed by exposure to low light levels exhibited less extensive induced chemical changes when challenge inoculated withL. terebrantis than did seedlings growing under higher light. Stem phloem tissue in these seedlings contained lower concentrations of-pinene than did nonstressed seedlings also challenge inoculated withL. terebrantis. It is hypothesized that monoterpenes and phenolics play a role in the defensive response of red pine against insect-fungal attack, that stress may predispose red pine to attack by insect-fungal complexes, and that such interactions are involved in red pine decline disease. Implications to plant defense theory and interactions among multiple stress agents in forest decline are discussed.To whom correspondence should be addressed at USDA Forest Service, 2500 Shreveport Hwy., Pineville, Louisiana 71360.     

Drought Stress Alters the Concentration of Wood Terpenoids in Scots Pine and Norway Spruce Seedlings

Drought is known to have an impact on the resistance of conifers to various pests, for example, by affecting resin flow in trees. Little is known, however, about the quantitative and qualitative changes in resin when trees are growing in low moisture conditions. We exposed Scots pine (Pinus sylvestris L.) and Norway spruce (Picea abies (L.) Karst.) seedlings to medium and severe drought stress for two growing seasons and analyzed the monoterpenes and resin acids in the main stem wood after two years of treatment. In addition to secondary chemistry, we measured the level of nutrients in the needles and the growth response of seedlings. After the first year of treatment, drought stress did not affect the growth of seedlings, but in the second year, shoot growth was retarded, especially in Scots pine. In both conifer species, severe drought increased the concentrations of several individual monoterpenes and resin acids. Total monoterpenes and resin acids were 39 and 32% higher in severe drought-treated Scots pine seedlings than in the controls, and 35 and 45% higher in Norway spruce seedlings. In Scots pine needles, the concentrations of nitrogen and phosphorus increased, while magnesium and calcium decreased compared to controls. In Norway spruce needles, nutrient concentrations were not affected. The results suggest that drought stress substantially affects both the growth of conifers and the chemical quality of the wood. We discuss the potential trade-off in growth and defense of small conifer seedlings.     

Herbivore-Induced Volatiles in the Perennial Shrub, <Emphasis Type="Italic">Vaccinium corymbosum</Emphasis>, and Their Role in Inter-branch Signaling

Herbivore feeding activates plant defenses at the site of damage as well as systemically. Systemic defenses can be induced internally by signals transported via phloem or xylem, or externally transmitted by volatiles emitted from the damaged tissues. We investigated the role of herbivore-induced plant volatiles (HIPVs) in activating a defense response between branches in blueberry plants. Blueberries are perennial shrubs that grow by initiating adventitious shoots from a basal crown, which produce new lateral branches. This type of growth constrains vascular connections between shoots and branches within plants. While we found that leaves within a branch were highly connected, vascular connectivity was limited between branches within shoots and absent between branches from different shoots. Larval feeding by gypsy moth, exogenous methyl jasmonate, and mechanical damage differentially induced volatile emissions in blueberry plants, and there was a positive correlation between amount of insect damage and volatile emission rates. Herbivore damage did not affect systemic defense induction when we isolated systemic branches from external exposure to HIPVs. Thus, internal signals were not capable of triggering systemic defenses among branches. However, exposure of branches to HIPVs from an adjacent branch decreased larval consumption by 70% compared to those exposed to volatiles from undamaged branches. This reduction in leaf consumption did not result in decreased volatile emissions, indicating that leaves became more responsive to herbivory (or “primed”) after being exposed to HIPVs. Chemical profiles of leaves damaged by gypsy moth caterpillars, exposed to HIPVs, or non-damaged controls revealed that HIPV-exposed leaves had greater chemical similarities to damaged leaves than to control leaves. Insect-damaged leaves and young HIPV-exposed leaves had higher amounts of endogenous cis-jasmonic acid compared to undamaged and non-exposed leaves, respectively. Our results show that exposure to HIPVs triggered systemic induction of direct defenses against gypsy moth and primed volatile emissions, which can be an indirect defense. Blueberry plants appear to rely on HIPVs as external signals for inter-branch communication.     

Terpene Attractant Candidates of Dioryctria sylvestrella in Maritime Pine (Pinus pinaster) Oleoresin, Needles, Liber, and Headspace Samples

Capillary GC analysis was used to determine the proportion and quantity of terpenes in wood resin, pentane extracts from needles and liber, and headspace samples of needles and pruning wounds in 24 thirteen-year-old maritime pine. Fifteen different terpenes were identified in the samples. Germacrene D and -pinene were the dominant terpenic compounds in the needles, while -pinene and -pinene were dominant in the liber and wood resin. Headspace samples of both needles and pruning wounds contained essentially monoterpenes. Only trace amounts of sesquiterpenes were found in pruning wound emissions. The presence of an oxygenated compound, linalool, in the pruning wound emissions is discussed, although this compound is not found in Maritime pine essential oil. Twelve of the 24 trees studied were infested by Dioryctria sylvestrella. Maritime pine susceptibility to this insect was related to the terpene composition of the different samples.     

Patterns of Monoterpene Variation within Individual Trees in Ponderosa Pine

We surveyed variation in both the concentration and composition of monoterpenes in six tissues within individuals of ponderosa pine to determine (1) whether variation exists between different tissues; (2) whether variation occurs between samples from the north and south sides of the tree within a tissue, and (3) whether composition of one tissue is correlated with that of others. Wood, roots, and resin exuded from the trunk have similar monoterpene compositions within trees. Phloem and cones contain a higher proportion of -pinene with less carene than resin, while needles are distinguished by high proportions of -pinene. Samples of the same tissue taken from the north and south sides of the tree differed little and were strongly correlated. Correlations were moderate across tissues within the groups sharing similar monoterpene compositions (e.g., phloem vs. cones), but correlations between tissues in different groups were weak (e.g., phloem vs. needles).     

Defensive mechanisms of loblolly and shortleaf pine against attack by southern pine beetle,Dendroctonus frontalis Zimmermann,and its fungal associate,Ceratocystis minor (Hedgecock) Hunt

Loblolly and shortleaf pine growing on a single site in the North Carolina piedmont were examined to determine similarities and differences in their defensive mechanisms against the southern pine beetle,Dendroctonus frontalis Zimmermann, and its fungal associate,Ceratocystis minor (Hedgecock) Hunt. Both species responded to wounding and fungal inoculation by forming a hypersensitive lesion around the wound site. There were significantly less soluble sugars and more monoterpenes in the lesion tissue than in unwounded inner bark. The two species were similar in resin flow rate and inner bark soluble sugar content, but the loblolly pines had thicker bark, longer hypersensitive lesions, and a higher concentration of inner bark monoterpenes. Inner bark monoterpene composition was also significantly different between the two pine species. It is hypothesized that two different defensive strategies against southern pine beetle attack may be utilized.     

Effects of nitrogen fertilization on secondary chemistry and ectomycorrhizal state of Scots pine seedlings and on growth of grey pine aphid

Effects of nitrogen availability on secondary compounds, mycorrhizal infection, and aphid growth of 1-year-old Scots pine (Pinus sylvestris L.) seedlings were studied during one growing season. Seedlings were fertilized with nutrient solutions containing low, optimum, and two elevated (2 × and 4 × optimum) levels of NH₄NO₃. At the end of growing season foliar nitrogen concentration, needle biomass, needle length, water contents of needles, root collar diameter, and number of buds increased with enhanced nitrogen availability. Addition of nitrogen did not have effect on concentrations of monoterpenes in growing needles, but in mature needles significantly decreased concentrations of some individual and total monoterpenes were detected. In growing needles the concentrations of some individual resin acids decreased, and in mature needles concentrations of some individual and total resin acids increased with increased nitrogen fertilization. Higher numbers of resin ducts were found in mature needles with nitrogen fertilization. Nitrogen fertilization decreased total phenolic concentrations in growing and mature needles of the current year, but in needles of the previous year no significant differences occurred. Mycorrhizal infection was highest at medium (optimum and 2 × optimum) nitrogen fertilization levels. The relative growth rate (RGR) of grey pine aphid [Schizolachnus pineti (F.)] responded positively to the increase in foliar nitrogen content. However, the increase in aphid performance between optimum and the highest fertilization level was slight. This may indicate a deterring effect of resin acids on aphids. The results indicate that carbon/nutrient balance hypothesis fails to predict directly the effects of nitrogen availability on concentrations of carbon-based defensive compounds in mature foliage. Altered nitrogen supply affects allocation to secondary metabolites differently, depending on the developmental state of the plant and the biosynthesis pathway, cost of synthesis, and storage of compounds.     

Feeding response of Ips paraconfusus to phloem and phloem metabolites of Heterobasidion annosum-inoculated ponderosa pine,Pinus ponderosa

In studies of feeding by the bark beetle, Ips paraconfusus, two pine stilbenes (pinosylvin and pinosylvin methyl ether), ferulic acid glucoside, and enantiomers of the four most common sugars present in ponderosa pine phloem (sucrose, glucose, fructose, and raffinose) did not stimulate or reduce male feeding when assayed on wet -cellulose with or without stimulatory phloem extractives present. When allowed to feed on wet -cellulose containing sequential extracts (hexane, methanol, and water) of ponderosa pine phloem, methanol and water extractives stimulated feeding, but hexane extractives did not. Males confined in wet -cellulose containing aqueous or organic extracts of culture broths derived from phloem tissue and containing the root pathogen, Heterobasidion annosum, ingested less substrate than beetles confined to control preparations. In an assay using logs from uninoculated ponderosa pines, the mean lengths of phloem in the digestive tracts increased as time spent feeding increased. Males confined to the phloem of basal logs cut from ponderosa pines artificially inoculated with H. annosum ingested significantly less phloem than beetles in logs cut from trees that were (combined) mock-inoculated or uninoculated and did not contain the pathogen. However, individual pathogen-containing treatments were not significantly different from uninoculated controls. It was concluded that altered feeding rates are not a major factor which may explain why diseased ponderosa pines are colonized by I. paraconfusus.     

Variation of Piperidine Alkaloids in Ponderosa (Pinus poderosa) and Lodgepole Pine (P. contorta) Foliage from Central Oregon

We quantified 2,6-disubstituted piperidine alkaloids in Pinus ponderosa and P. contorta needles from three forest sites in April, June, August, and December. Alkaloids were detected on at least one date in 71% of the ponderosa pine and in 29% of the lodgepole pine trees sampled. Pinidine was the major alkaloid constituent of ponderosa pine, while euphococcinine was the predominant compound in lodgepole pine. For ponderosa pine, total alkaloid concentrations were very low at two sites on all dates. At the third site, concentrations were variable but significantly higher on all dates. Total alkaloid concentrations in previous-year foliage from this site were highest in April, then significantly lower from June through December. Current-year foliage collected in August and December had significantly higher alkaloid concentrations than previous-year foliage on the same dates. Variation in foliar nitrogen concentrations accounted for some of the alkaloid variation in current-year foliage sampled in August.     

Volatile Emissions Triggered by Multiple Herbivore Damage: Beet Armyworm and Whitefly Feeding on Cotton Plants

Plants are commonly attacked by more than one species of herbivore, potentially causing the induction of multiple, and possibly competing, plant defense systems. In the present paper, we determined the interaction between feeding by the phloem feeder silverleaf whitefly (SWF), Bemisia tabaci Gennadius (B-biotype = B. argentifolii Bellows and Perring), and the leaf-chewing beet armyworm (BAW), Spodoptera exigua Hübner, with regard to the induction of volatile compounds from cotton plants. Compared to undamaged control plants, infestation with SWF did not induce volatile emissions or affect the number and density of pigment glands that store volatile and nonvolatile terpenoid compounds, whereas infestation by BAW strongly induced plant volatile emission. When challenged by the two insect herbivores simultaneously, volatile emission was significantly less than for plants infested with only BAW. Our results suggest that tritrophic level interactions between cotton, BAW, and natural enemies of BAW, that are known to be mediated by plant volatile emissions, may be perturbed by simultaneous infestation by SWF. Possible mechanisms by which the presence of whiteflies may attenuate volatile emissions from caterpillar-damaged cotton plants are discussed.     

Plant virus-induced changes in aphid population development and temporal fluctuations in plant nutrients

Cucurbita pepo plants were infected with zucchini yellow mosaic virus or maintained noninfected.Aphis gossypii, which transmits the virus, lived longer and produced more offspring on infected than on noninfected plants. On infected plants, the intrinsic rate of natural increase forA. gossypii increased with time after inoculation. In a similar experiment, concentrations of phloem sap nutrients, including free amino acids, total protein, and sugars from infected and noninfected plants were compared for 37 days after inoculation. Significant differences in levels of individual amino acids from phloem exudate between infected and noninfected plants were found, yet the concentration of total amino acids was not substantially different between infected and noninfected plants. Beginning four days after inoculation, the total protein content of phloem exudate generally was lower in infected plants than noninfected plants. Likewise, the total sugar content of phloem exudate from infected plants was lower than that of noninfected plants beginning nine days after inoculation. In contrast with the results from analyses of phloem exudate, foliage from infected plants had higher levels of almost all amino acids than noninfected foliage beginning nine days after inoculation. Concentrations of individual and total amino acids in infected foliage increased throughout the experimental period. Although no temporal effects were observed in the foliage sugar content for either individual or total sugars, starch content decreased with time in infected plants, while in noninfected plants, starch content remained level.     

Preference of meadow voles (Microtus pennsylvanicus) for conifer seedlings: Chemical components and nutritional quality of bark of damaged and undamaged trees

Meadow voles (Microtus pennsylvanicus Ord.) of the 1987–1988 population density peak left several seedling plantations of Norway spruce (Picea abies) and Norway pine (Pinus resinosa) severely damaged in southern Quebec, Canada, while white spruce (Picea glauca) and white pine (Pinus strobus) suffered no damage. We compared levels of crude protein, total nonstructural carbohydrates (TNC), total phenols, and monoterpenes in the bark to detect differences between damaged and undamaged seedlings within and between plantations. There were no significant differences in levels of nutritional components between both types of seedlings within the same plantation. However, differences were noted among tree plantations for protein, TNC, and phenolics content but these differences could not be assigned to the voles' selection for particular species. Quantitative and qualitative differences also were registered in monoterpene levels between species. -Myrcene and bornyl acetate were found only in seedlings of undamaged species. Moreover, the latter contained higher levels of limonene. We conclude that the presence of specific monoterpenes is more important in defense mechanisms of conifer seedlings than relative levels of nutrients (protein or TNC) or total phenolics.     

Variation in Growth, Chemical Defense, and Herbivore Resistance in Scots Pine Provenances

Variation in the growth, chemical defense, and susceptibility to monophagous and polyphagous sucking herbivores was assessed for four Scots pine (Pinus sylvestris) provenances in Petri dish and pot experiments. Seed provenances, Muonio (MU), Suomussalmi (SU), Korpilahti (KO), and Saaremaa (SA) represented a 1200-km N–S gradient. Scots pines from SU were least preferred as a host plant by pine-feeding aphids Schizolachnus pineti Fabr. and Eulachnus agilis, and in pot experiments, pines from SA were most preferred. S. pineti also produced the fewest nymphs on pines from SU. Polyphagous Lygus rugulipennis laid significantly fewer eggs on pines from MU provenance, which were smaller than seedlings from other provenances. The total and some individual terpene concentrations increased, while the total resin acid concentration decreased, towards the north. The absolute concentrations of total or individual terpenes and resin acids in the needles of unfavored SU plants did not differ significantly from the other provenances. Only the proportional quantity of dehydroabietic acid was higher in SU plants than in KO plants, and it correlated, together with sandaracopimaric acid, negatively with the number of S. pineti nymphs in the reproduction experiment. Conversely, absolute concentrations of some terpenes correlated negatively and some resin acids positively with the number of S. pineti nymphs. The length and weight of shoots decreased towards the north, adequately explaining the number of Lygus eggs and the final numbers of both aphids. The results suggest that seedling size is a better estimator of susceptibility of pine seedlings to both specialist and generalist sucking insects than relative or absolute concentrations of monoterpenes and resin acids.     

Weak responses of pine sawfly larvae to high needle flavonoid concentrations in scots pine

Responses of sawfly larvae (Hymenoptera, Diprionidae) to the flavonoid taxifolin glucoside in their host plant were studied in a laboratory experiment. Larvae ofNeodiprion sertifer andDiprion pini were raised from egg hatch to cocoon spinning on two Scots pine (Pinus sylvestris) chemotypes, one without needle taxifolin glucoside (–) and the other containing 2–4% taxifolin glucoside (+). The (+) chemotype had somewhat lower concentrations of needle terpenoids (resin acids) than the (–) chemotype. Current-year needles had higher taxifolin glucoside concentrations than mature needles. There were no differences in survival or body size betweenN. sertifer larvae that fed on the (+) chemotype and those that fed on the (–) chemotype. FemaleD. pini larvae raised on (+) needles developed 6% more slowly than larvae fed (–) needles. The results from this study are contrary to earlier findings showing that flavonoid glucosides have strong negative effects on insect performance. Possible explanations for the different outcomes are discussed.     

Effects of Light and Nutrient Availability on Aspen: Growth, Phytochemistry, and Insect Performance

This study explored the effect of resource availability on plant phytochemical composition within the framework of carbon–nutrient balance (CNB) theory. We grew quaking aspen (Populus tremuloides) under two levels of light and three levels of nutrient availability and measured photosynthesis, productivity, and foliar chemistry [water, total nonstructural carbohydrates (TNC), condensed tannins, and phenolic glycosides]. Gypsy moths (Lymantria dispar) and forest tent caterpillars (Malacosoma disstria) were reared on foliage from each of the treatments to determine effects on insect performance. Photosynthetic rates increased under high light, but were not influenced by nutrient availability. Tree growth increased in response to both the direct and interactive effects of light and nutrient availability. Increasing light reduced foliar nitrogen, while increasing nutrient availability increased foliar nitrogen. TNC levels were elevated under high light conditions, but were not influenced by nutrient availability. Starch and condensed tannins responded to changes in resource availability in a manner consistent with CNB theory; levels were highest under conditions where tree growth was limited more than photosynthesis (i.e., high light–low nutrient availability). Concentrations of phenolic glycosides, however, were only moderately influenced by resource availability. In general, insect performance varied relatively little among treatments. Both species performed most poorly on the high light–low nutrient availability treatment. Because phenolic glycosides are the primary factor determining aspen quality for these insects, and because levels of these compounds were minimally affected by the treatments, the limited response of the insects was not surprising. Thus, the ability of CNB theory to accurately predict allocation to defense compounds depends on the response of specific allelochemicals to changes in resource availability. Moreover, whether allelochemicals serve to defend the plant depends on the response of insects to specific allelochemicals. Finally, in contrast to predictions of CNB theory, we found substantial allocation to storage and defense compounds under conditions in which growth was carbon-limited (e.g., low light), suggesting a cost to defense in terms of reduced growth.     

Role of phenolics of coniferous trees as deterrents against debarking behavior of meadow voles (Microtus pennsylvanicus)

Preferences of meadow voles (Microtus pennsylvanicus) toward various coniferous species were tested under winter conditions. Cafeteria tests showed preferences for Norway spruce (Picea abies) and Norway pine (Pinus resinosa) and rejection of white pine (Pinus strobus) and white spruce (Picea glauca). When phenolic extracts of these species were prepared and added to a low 8% protein diet, food intake of voles and preferences by rank followed the same patterns. Determination of the concentration of phenolics in branch samples using the Singleton and Rossi procedure showed that Norway pines contained the smallest amount (2.15%/g dry matter) while spruces yielded the most (2.76%/g dry matter). These experiments lead us to expect high losses of Norway pine and Norway spruce in habitats harboring high vole densities.     

Fertility,Root Reserves and the Cost of Inducible Defenses in the Perennial Plant <Emphasis Type="Italic">Solanum carolinense</Emphasis>

We examined the relationship between internal resources (root reserves), external resources (soil fertility), and allocation to defense vs. growth in the clonal, perennial herb Solanum carolinense. In a short-term (9 d) greenhouse experiment, plants were treated once with jasmonic acid (JA) to determine if polyphenols and glycoalkaloids were inducible by simulated herbivory. In a longer-term (4 wk) greenhouse experiment, we measured the cost, in terms of growth, of treatment with JA every 3 d, to determine if the induced response was due more to carbon limitation or nitrogen limitation. We manipulated the resources available to the plants by varying soil fertility and the size of root cuttings from which plants were grown, and assessed how different resource levels affected the growth and production of polyphenols and alkaloids under JA treatment or control conditions. In the short term, JA increased the concentration of polyphenols in both above- and belowground plant parts, as well as alkaloid concentrations in the roots. In the long term, the only significant secondary chemistry response to JA was an increased polyphenol concentration in above ground tissues. The total amount of polyphenols produced was the same for JA and control plants, indicating that the higher concentration was a result of the lower biomass of treated plants. In contrast, alkaloid concentrations in plants treated with JA for 4 wk did not differ from controls, but JA-treated plants contained lower total amounts of alkaloids in above ground tissues, as a result of decreased growth. Fertilizer level and root cutting size had effects on growth and the production of secondary compounds and influenced the cost of induction. Plants grown under high fertility had a greater reduction in growth in response to JA than plants grown under low fertility, indicating a greater trade-off between growth and defense for high fertility plants. Plants from larger root cuttings grew bigger without any reduction in the concentration of polyphenols and alkaloids. We demonstrated that the phenotype of S. carolinense was plastic in response to simulated herbivory, fertility level, and root cutting size, and that there was a significant growth cost to induction that varied with the environment and appears to be due in large part to the allocation of limited carbon reserves.     

Effect of nitrogen and water treatment on leaf chemistry in horsenettle (Solanum carolinense), and relationship to herbivory by flea beetles (Epitrix spp.) and tobacco hornworm (Manduca sexta)

We studied the interaction between plants (horsenettle; Solanum carolinense) and herbivorous insects (flea beetles; Epitrix spp., and tobacco hornworm; Manduca sexta) by focusing on three questions: (1) Does variation in nitrogen availability affect leaf chemistry as predicted by the carbon-nutrient balance (CNB) hypothesis? (2) Does variation in plant treatment and leaf chemistry affect insect feeding? (3) Is there an interaction between the insect herbivores that is mediated by variation in leaf chemistry? For three successive years (1998-2001), we grew a set of clones of 10 maternal plants under two nitrogen treatments and two water treatments. For each plant in the summer of 2000, we assayed herbivory by hornworms in both indoor (detached leaf) and outdoor (attached leaf) assays, as well as ambient flea beetle damage. Estimates of leaf material consumed were made via analysis of digitized leaf images. We also assayed leaves for total protein, phenolic, and glycoalkaloid content, and for trypsin inhibitor, polyphenol oxidase, and peroxidase activity. Despite strong effects of nitrogen treatment on growth and reproduction, only total protein responded as predicted by CNB. Leaf phenolic levels were increased by nitrogen treatment, polyphenol oxidase activity was decreased, and other leaf parameters were unaffected. Neither hornworm nor flea beetle herbivory could be related to plant treatment or genotype or to variation in any of the six leaf chemical parameters. A negative relationship between flea beetle and hornworm herbivory was found, but was not apparently mediated by any of the measured leaf chemicals. Because leaf resistance was maintained in low nitrogen plants at the apparent expense of growth and reproduction, our results support the concept of a fitness cost of defense, as predicted by the optimal defense hypothesis.     

Phenolic Chemistry of Coast Live Oak Response to <Emphasis Type="Italic">Phytophthora ramorum</Emphasis> Infection

Since the mid 1990s, Phytophthora ramorum has been responsible for the widespread mortality of tanoaks, as well as several oak species throughout California and Oregon forests. However, not all trees die, even in areas with high disease pressure, suggesting that some trees may be resistant to the pathogen. In this study, the chemical basis of host resistance was investigated. Three field experiments were carried out in California between December 2004 and September 2005. The levels of nine phenolic compounds (gallic acid, catechin, tyrosol, a tyrosol derivative, ellagic acid, and four ellagic acid derivatives) extracted from the phloem of trees that had been either artificially inoculated with P. ramorum or trees putatively infected with P. ramorum (based on canker symptoms) were quantified by high-performance liquid chromatography (HPLC). Significant differences in phenolic profiles were found between phloem sampled from the active margins of cankers, healthy phloem from asymptomatic trees, and phloem sampled 60 cm away from canker sites, although the magnitude and direction of the responses was not consistent across all experiments. Concentrations of gallic acid, tyrosol, and ellagic acid showed the greatest differences in these different tissues, but varied considerably across treatments. Gallic acid and tyrosol were tested in in vitro bioassays and showed strong dose-dependent inhibitory effects against P. ramorum, P. cinnamomi, P. citricola, and P. citrophthora. These results suggest that phloem chemistry varies in response to pathogen infection in California coast live oak populations and that changes in phloem chemistry may be related to apparently resistant phenotypes observed in the field. Frances S. Ockels and Alieta Eyles contributed equally to the paper