Yellow-Cedar, Callitropsis (Chamaecyparis) nootkatensis, Secondary Metabolites, Biological Activities, and Chemical Ecology.

Yellow-cedar, Callitropsis nootkatensis, is prevalent in coastal forests of southeast Alaska, western Canada, and inland forests along the Cascades to northern California, USA. These trees have few microbial or animal pests, attributable in part to the distinct groups of biologically active secondary metabolites their tissues store for chemical defense. Here we summarize the new yellow-cedar compounds identified and their biological activities, plus new or expanded activities for tissues, extracts, essential oils and previously known compounds since the last review more than 40 years ago. Monoterpene hydrocarbons are the most abundant compounds in foliage, while heartwood contains substantial quantities of oxygenated monoterpenes and oxygenated sesquiterpenes, with one or more tropolones. Diterpenes occur in foliage and bark, whereas condensed tannins have been isolated from inner bark. Biological activities expressed by one or more compounds in these groups include fungicide, bactericide, sporicide, acaricide, insecticide, general cytotoxicity, antioxidant and human anticancer. The diversity of organisms impacted by whole tissues, essential oils, extracts, or individual compounds now encompasses ticks, fleas, termites, ants, mosquitoes, bacteria, a water mold, fungi and browsing animals. Nootkatone, is a heartwood component with sufficient activity against arthropods to warrant research focused toward potential development as a commercial repellent and biopesticide for ticks, mosquitoes and possibly other arthropods that vector human and animal pathogens.

Ethanol attracts scolytid beetles to Phytophthora ramorum cankers on coast live oak.

Ethanol in sapwood was analyzed along vertical transects, through small spot cankers and larger basal cankers, of Phytophthora ramorum-infected stems of Quercus agrifolia at three sites in California. Trees with large basal cankers, known to attract scolytid beetles, had a 4.3 times higher ethanol level than trees with spot cankers that attract fewer beetles. Ethanol concentrations inside cankers, where scolytid beetles preferentially attack, varied by about four orders of magnitude among samples, with a median level of 16.0 µg.g(-1) fresh mass. This concentration was 4.3 and 15.5 times greater, respectively, than the concentrations at 1 cm or 15-30 cm outside the canker boundaries. In the laboratory, we demonstrated that ethanol escaped through the bark of a Q. garryana log just 3 days after it was added to the sapwood. At the three study sites, traps baited with ethanol captured more Xyleborinus saxesenii, Pseudopityophthorus pubipennis, and Monarthrum dentiger (all Coleoptera: Curculionidae: Scolytinae) than traps baited with ethanol plus (-)-α-pinene, or ethanol plus 4-allylanisole (4AA). Logs of Q. agrifolia with a 50 % ethanol solution added to the sapwood were placed at the study sites, with or without additional bark treatments above the ethanol. The number of scolytid beetle gallery holes above the ethanol-infused sapwood was 4.4 times greater than that on the opposite side of the log where no ethanol was added. Attachment of ultra-high release (-)-α-pinene pouches to the bark surface above the 50 % ethanol solution reduced scolytid attacks to a density of 19.1 % that of logs without this treatment. We conclude that ethanol in P. ramorum cankers functions as a primary host attractant for scolytid beetles and is an important link in colonization of these cankers and accelerated mortality of Q. agrifolia. The results of this research shed light on the chemical ecology behind the focused scolytid attacks on P. ramorum-infected coast live oaks, and lay the groundwork for future efforts to prolong the survival of individual trees of this keystone species.

Effect of plant sterols and tannins on Phytophthora ramorum growth and sporulation.

Elicitin-mediated acquisition of plant sterols is required for growth and sporulation of Phytophthora spp. This study examined the interactions between elicitins, sterols, and tannins. Ground leaf tissue, sterols, and tannin-enriched extracts were obtained from three different plant species (California bay laurel, California black oak, and Oregon white oak) in order to evaluate the effect of differing sterol/tannin contents on Phytophthora ramorum growth. For all three species, high levels of foliage inhibited P. ramorum growth and sporulation, with a steeper concentration dependence for the two oak samples. Phytophthora ramorum growth and sporulation were inhibited by either phytosterols or tannin-enriched extracts. High levels of sterols diminished elicitin gene expression in P. ramorum; whereas the tannin-enriched extract decreased the amount of 'functional' or ELISA-detectable elicitin, but not gene expression. Across all treatment combinations, P. ramorum growth and sporulation correlated strongly with the amount of ELISA-detectable elicitin (R (2) = 0.791 and 0.961, respectively).

Red turpentine beetle primary attraction to (-)-ß-pinene+ethanol in US Pacific Northwest ponderosa pine forests.

Red turpentine beetle, Dendroctonus valens (Coleoptera: Curculionidae: Scolytinae) is a non-aggressive pine bark beetle native to North America, and more aggressive invader in China. Dispersing pioneer beetles are attracted to potential host trees by oleoresin monoterpene kairomones, but respond more strongly to those combined with ethanol, a mixture often released from stressed, dying, or recently dead trees. (+)-3-Carene, usually the dominant or co-dominant monoterpene in ponderosa pine, Pinus ponderosa, is a stronger attractant than α-pinene or ß-pinene where tested over a large portion of the D. valens range, while (+)-3-carene+ethanol was shown previously to attract twice the beetles of (+)-3-carene. A field test comparing D. valens attraction among the three monoterpenes when all are released with ethanol has never been reported, and was our objective. In three US Pacific Northwestern pine forests, (-)-ß-pinene+ethanol lures attracted 1.4 to 1.9 times more beetles than (+)-3-carene+ethanol. (+)- or (±)-α-pinene+ethanol lures were least attractive. A 1:1:1 monoterpene mixture+ethanol lure attracted more beetles than the 1:1:1 lure, but it was not statistically higher. Monoterpenes were dispensed from low density polyethylene bottles and their release rates monitored in laboratory and field tests. Under laboratory conditions (+)-3-carene was released much more rapidly than (+)-α-pinene or (-)-ß-pinene when dispensed separately, or in a 1:1:1 mixture. (+)-3-Carene in the 1:1:1 mixture increased the release of both pinenes over their rates when dispensed separately. (-)-ß-Pinene+ethanol is currently the strongest kairomone lure for D. valens attraction in US northwest pine forests, and has value for beetle detection, monitoring, research, and management.

Genetic variation of piperidine alkaloids in Pinus ponderosa: a common garden study.

BACKGROUND AND AIMS: Previous measurements of conifer alkaloids have revealed significant variation attributable to many sources, environmental and genetic. The present study takes a complementary and intensive, common garden approach to examine genetic variation in Pinus ponderosa var. ponderosa alkaloid production. Additionally, this study investigates the potential trade-off between seedling growth and alkaloid production, and associations between topographic/climatic variables and alkaloid production. METHODS: Piperidine alkaloids were quantified in foliage of 501 nursery seedlings grown from seed sources in west-central Washington, Oregon and California, roughly covering the western half of the native range of ponderosa pine. A nested mixed model was used to test differences among broad-scale regions and among families within regions. Alkaloid concentrations were regressed on seedling growth measurements to test metabolite allocation theory. Likewise, climate characteristics at the seed sources were also considered as explanatory variables. KEY RESULTS: Quantitative variation from seedling to seedling was high, and regional variation exceeded variation among families. Regions along the western margin of the species range exhibited the highest alkaloid concentrations, while those further east had relatively low alkaloid levels. Qualitative variation in alkaloid profiles was low. All measures of seedling growth related negatively to alkaloid concentrations on a natural log scale; however, coefficients of determination were low. At best, annual height increment explained 19.4 % of the variation in ln(total alkaloids). Among the climate variables, temperature range showed a negative, linear association that explained 41.8 % of the variation. CONCLUSIONS: Given the wide geographic scope of the seed sources and the uniformity of resources in the seedlings' environment, observed differences in alkaloid concentrations are evidence for genetic regulation of alkaloid secondary metabolism in ponderosa pine. The theoretical trade-off with seedling growth appeared to be real, however slight. The climate variables provided little evidence for adaptive alkaloid variation, especially within regions.

Photosynthetic Declines in Phytophthora ramorum-Infected Plants Develop Prior to Water Stress and in Response to Exogenous Application of Elicitins.

ABSTRACT Phytophthora ramorum, causal agent of sudden oak death, is responsible for widespread oak mortality in California and Oregon, and has the potential to infect 100 or more species. Symptoms range from stem girdling and shoot blight to leaf spotting. In this study, we examined the physiological impacts of P. ramorum infection on Rhododendron macrophyllum. In stem-inoculated plants, photosynthetic capacity (V(cmax)) significantly declined by approximately 21% 3 weeks after inoculation in visibly asymptomatic leaves. By 4 weeks, after the development of significant stem lesions and loss in water transport capacity, water stress led to stomatal closure and additional declines in photosynthetic capacity. We also report the isolation, characterization, and biological activity of two P. ramorum elicitins. Both elicitins were capable of inducing a hypersensitive-like response in one incompatible (Nicotiana tabacum SR1) and three compatible hosts (R. macrophyllum, Lithocarpus densiflorus, and Umbellularia californica). Infiltration of leaves from all three compatible hosts with both P. ramorum elicitins caused significant declines in chlorophyll fluorescence (F(v) /F(m)). For all four species, the loss of photosynthetic capacity was directly proportional to H(+) uptake and ethylene production, two common components of the hypersensitive response. This is the first report of elicitins causing photosynthetic declines in compatible hosts independent of plant water stress.

Biological screening of selected Pacific Northwest forest plants using the brine shrimp (Artemia salina) toxicity bioassay.

The brine shrimp (Artemia salina) bioassay was used to screen 211 methanol extracts from 128 species of Pacific Northwest plants in search of general cytotoxic activity. Strong toxicity (LC50 < 100 µg/ml) was found for 17 extracts from 13 species, with highest activity observed for Angelica arguta roots at <10 µg/ml. Notably, four species of cedar trees and one of juniper in the family Cupressaceae dominated this group with LC50 for heartwood extracts ranging from 15 to 89 µg/ml. Moderate toxicity (LC50 100-500 µg/ml) was found in 38 extracts from 27 species, while weak toxicity (LC50 500-1000 µg/ml) was detected for 17 extracts in 16 species. There were 139 extracts from 99 species that were non-toxic (LC50 > 1000 µg/ml). Our subsequent studies of conifer heartwoods with strong activity confirm the assay's value for identifying new investigational leads for materials with insecticidal and fungicidal activity.

Piperidine alkaloids in sitka spruce with varying levels of resistance to white pine weevil (Coleoptera: Curculionidae).

Our objective was to evaluate piperidine alkaloids as potential resistance factors in Sitka spruce, Picea sitchensis (Bong.) Carr, at risk to attack by white pine weevils, Pissodes strobi (Peck). We sampled 72 seedlings in each of two replicated field trials in the Oregon Coast Range. The seedlings were grown from open-pollinated seeds of putatively "resistant" or "susceptible" off-site parental sources. Alkaloid concentrations in bark and foliage were measured in previously unattacked trees at the time of weevil host selection. Leader mortality was evaluated in the fall to gauge actual resistance in the sample trees. Five families had < or = 25% topkill and seven sustained >50% topkill. Alkaloid concentrations differed significantly among families, but the major alkaloids did not appear to be functionally linked with topkill or useful indicators of resistance. However, our study design did not address all potential resistance mechanisms. Therefore, before concluding that Sitka spruce alkaloids have no influence on white pine weevils, complementary laboratory and field experiments are needed.

Plant protein and secondary metabolites influence diet selection in a mammalian specialist herbivore.

For herbivores, nutrient intake is limited by the relatively low nutritional quality of plants and high concentrations of potentially toxic defensive compounds (plant secondary metabolites, PSMs) produced by many plants. In response to phytochemical challenges, some herbivores selectively forage on plants with higher nutrient and lower PSM concentrations relative to other plants. Pygmy rabbits (Brachylagus idahoensis) are dietary specialists that feed on sagebrush (Artemisia spp.) and forage on specific plants more than others within a foraging patch. We predicted that the plants with evidence of heavy foraging (browsed plants) would be of higher dietary quality than plants that were not browsed (unbrowsed). We used model selection to determine which phytochemical variables best explained the difference between browsed and unbrowsed plants. Higher crude protein increased the odds that plants would be browsed by pygmy rabbits and the opposite was the case for certain PSMs. Additionally, because pygmy rabbits can occupy foraging patches (burrows) for consecutive years, their browsing may influence the nutritional and PSM constituents of plants at the burrows. In a post hoc analysis, we did not find a significant relationship between phytochemical concentrations, browse status and burrow occupancy length. We concluded that pygmy rabbits use nutritional and chemical cues while making foraging decisions.

Two new diterpene phenols from Calocedrus decurrans.

Two new p-cymene based diphenols (1-2) were isolated from the heartwood of Calocedrus decurrans. Structures were elucidated by 1D and 2D NMR techniques and HRMS. Libocedroquinone (3) was also isolated as a natural product for the first time. A new system of nomenclature is proposed for description of such oligomeric p-cymene derivatives.

Antimicrobial activity of extractable conifer heartwood compounds toward Phytophthora ramorum.

Ethyl acetate extracts from heartwood of seven western conifer trees and individual volatile compounds in the extracts were tested for antimicrobial activity against Phytophthora ramorum. Extracts from incense and western redcedar exhibited the strongest activity, followed by yellow-cedar, western juniper, and Port-Orford-cedar with moderate activity, and no activity for Douglas-fir and redwood extracts. Chemical composition of the extracts varied both qualitatively and quantitatively among the species with a total of 37 compounds identified by mass spectrometry. Of the 13 individual heartwood compounds bioassayed, three showed strong activity with a Log(10) EC(50) less than or equal to 1.0 ppm (hinokitiol, thymoquinone, and nootkatin), three expressed moderate activity ranging from 1.0-2.0 ppm (nootkatol, carvacrol, and valencene-11,12-diol), four compounds had weak activity at 2.0-3.0 ppm [alpha-terpineol, valencene-13-ol, (+)-beta-cedrene, (-)-thujopsene], and three had no activity [(+)-cedrol, delta-cadinene, and methyl carvacrol]. All of the most active compounds contained a free hydroxyl group, except thymoquinone. The importance of a free hydroxyl was demonstrated by the tremendous difference in activity between carvacrol (Log(10) EC(50) 1.81 +/- 0.08 ppm) and methyl carvacrol (Log(10) EC(50) >3.0 ppm). A field trial in California, showed that heartwood chips from redcedar placed on the forest floor for 4 months under Umbellularia californica (California bay laurel) with symptoms of P. ramorum leaf blight significantly limited the accumulation of P. ramorum DNA in the litter layer, compared with heartwood chips from redwood.

Changes in heartwood chemistry of dead yellow-cedar trees that remain standing for 80 years or more in southeast Alaska.

We measured the concentrations of extractable bioactive compounds in heartwood of live yellow-cedar (Chamaecyparis nootkatensis) trees and five classes of standing snags (1-5, averaging 4, 14, 26, 51, and 81 years-since-death, respectively) to determine how the concentrations changed in the slowly deteriorating snags. Three individuals from each of these six condition classes were sampled at four sites spanning a 260-km distance across southeast Alaska, and the influence of geographic location on heartwood chemistry was evaluated. Cores of heartwood were collected at breast height and cut into consecutive 5-cm segments starting at the pith. Each segment was extracted with ethyl acetate and analyzed by gas chromatography. Concentrations of carvacrol, nootkatene, nootkatol, nootkatone, nootkatin, and total extractives (a sum of 16 compounds) for the inner (0-5 cm from pith), middle (5-10 cm from pith), and surface (outer 1.1-6.0 cm of heartwood) segments from each core were compared within each tree condition class and within segments across condition classes. Heartwood of class 1 and 2 snags had the same chemical composition as live trees. The first concentration changes begin to appear in class 3 snags, which coincides with greater heartwood exposure to the external environment as decaying sapwood sloughs away, after losing the protective outer bark. Within core segments, the concentrations of all compounds, except nootkatene, decrease between snag classes 2 and 5, resulting in the heartwood of class 5 snags having the lowest quantities of bioactive compounds, although not different from the amounts in class 4 snags. This decline in chemical defense is consistent with heartwood of class 5 snags being less decay-resistant than heartwood of live trees, as observed by others. The unique heartwood chemistry of yellow cedar and the slow way it is altered after death allow dead trees to remain standing for up to a century with a profound impact on the ecology of forests in southeast Alaska where these trees are in decline.

Ethanol synthesis and aerobic respiration in the laboratory by leader segments of Douglas-fir seedlings from winter and spring.

Stem segments from terminal leaders of Douglas-fir, Pseudotsuga menziesii (Mirb.) Franco, seedlings were sampled in mid-December when cambial cells were dormant. The residual, debudded leaders were resampled again in early May when the cambium was metabolically active. May stems had higher constitutive ethanol concentrations than December stems. This was not the result of cambial hypoxia generated by rapid spring respiration rates, because when aerobic respiration was stimulated by incubating the stems in air at 30 degrees C ethanol production was induced in December, but not in May. Rapid respiration rates at 30 degrees C may have depleted O(2) supplies and induced ethanol production in December stems because dormant, thick-walled cambial cells may be less permeable to CO(2) and O(2), compared with metabolically active, thin-walled cambial cells in May. December stem segments incubated in a N(2) atmosphere at 30 degrees C synthesized 1.8 times more ethanol than segments from May, most likely because spring growth had reduced the soluble sugars available for fermentation. CO(2) efflux from May stems (after 5.5 h of incubation at 30 degrees C) was equal to December stems per unit volume, but greater than December stems per unit surface area. N(2)-induced ethanol concentrations were positively related with CO(2) efflux per unit volume, indicating that rapidly respiring leaders can maintain rapid fermentation rates, provided soluble sugars are readily available. N(2)-induced ethanol and CO(2) efflux per unit volume declined with increasing leader diameter in both seasons, whereas there were no relationships between CO(2) efflux per unit surface area and diameter. Cambium physiology and phenology influence the induction of fermentation and concentrations of ethanol produced in terminal leaders of Douglas-fir, and probably other conifers as well. This needs to be considered when comparing fermentation among species, or comparing individuals from different seasons, or disparate ages within a species.

Hydraulic conductivity in roots of ponderosa pine infected with black-stain (Leptographium wageneri) or annosus (Heterobasidion annosum) root disease.

Roots from healthy and diseased mature ponderosa pine, Pinus ponderosa Laws., trees were excavated from a site near Burns, Oregon. The diseased trees were infected with black-stain root disease, Leptographium wageneri Kendrick, or annosus root disease, Heterobasidion annosum (Fr.) Bref., or both. Axial hydraulic conductivity of the roots was measured under a positive head pressure of 5 kPa, and the conducting area was stained with safranin dye to determine specific conductivity (k(s)). In diseased roots, only 8-12% of the cross-sectional xylem area conducted water. Resin-soaked xylem completely restricted water transport and accounted for 13-16% of the loss in conducting area. In roots with black-stain root disease, 17% of the loss in conducting area was associated with unstained xylem, possibly resulting from occlusions or embolisms. Based on the entire cross-sectional area of infected roots, the k(s) of roots infected with black-stain root disease was 4.6% of that for healthy roots, whereas the k(s) of roots infected with annosus root disease was 2.6% of that for healthy roots. Although these low values were partly the result of the presence of a large number of diseased roots (72%) with no conducting xylem, the k(s) of functional xylem of diseased roots was only 33% of that for healthy roots. The low k(s) values of functional xylem in diseased roots may be caused by fungus induced occlusions preceding cavitation and embolism of tracheids. The k(s) of disease-free roots from diseased trees was only 70% of that for healthy roots from healthy trees. The disease-free roots had the same mean tracheid diameter and tissue density as the healthy roots, suggesting that the lower k(s) in disease-free roots of diseased trees may also have been caused by partial xylary occlusions.