Pine needle abortion in cattle: metabolism of isocupressic acid.

The rumen and hepatic metabolism of the cattle abortifacient compound isocupressic acid (ICA) was examined in vitro and in vivo. ICA was incubated for 56 h in bovine rumen inoculum and was found to be converted to three compounds identified as imbricatoloic acid, a structurally uncharacterized isomer of imbricatoloic acid, and dihydroagathic acid. In preparations of liver homogenates, ICA was found to be oxidized to agathic acid. No differences in ICA metabolites were detected in comparing the cow, sheep, pig, goat, guinea pig, and rat livers; however, guinea pig and rat liver homogenates were less efficient in converting ICA to agathic acid. ICA had been administered to cows orally and by intravenous infusion and induced abortions after either method of treatment. After intravenous infusion, agathic acid was identified as the major metabolite together with minor amounts of dihydroagathic acid. After oral administration, dihydroagathic acid was identified as the major metabolite with minor amounts of agathic acid, imbricatoloic acid, and a structurally uncharacterized metabolite tentatively identified as tetrahydroagathic acid.     

Timber harvesting alters soil carbon mineralization and microbial community structure in coniferous forests

Timber harvesting influences both above and belowground ecosystem nutrient dynamics. Impact of timber harvesting on soil organic matter (SOM) mineralization and microbial community structure was evaluated in two coniferous forest species, ponderosa pine (Pinus ponderosa) and lodgepole pine (Pinus contorta). Management of ponderosa pine forests, particularly even-aged stand practices, increased the loss of CO₂-C and hence reduced SOM storage potential. Changes in soil microbial community structure were more pronounced in ponderosa pine uneven-aged and heavy harvest stands and in lodgepole pine even-aged stand as compared to their respective unmanaged stands. Harvesting of trees had a negative impact on SOM mineralization and soil microbial community structure in both coniferous forests, potentially reducing coniferous forest C storage potential.     

Preparation of tetrahydroagathic acid: a serum metabolite of isocupressic acid, a cattle abortifacient in ponderosa pine

Isocupressic acid (1) was used to synthetically prepare a mixture of (8S,13R,S)-labda-15,19-dioic acid (tetrahydroagathic acid) (5) via a two-step oxidation procedure followed by hydrogenation of the double bonds at C13 and C8. Reduction of the C8,17 double bond was stereospecific producing only the 8S isomer and confirmed by the nOe interaction between the resulting C17 and C20 methyl groups. The 13R and 13S isomers of 5 were separated and analyzed by HPLC/MS, and (13S)-tetrahydroagathic acid was isolated and identified by comparison to a standard prepared by hydrogenation of naturally occurring (13S)-dihydroagathic acid (4). (13R,S)-dihydroagathic acid was prepared by selective sodium metal-catalyzed hydrogenation of the C13,14 allylic double bond of agathic acid (3). The prepared compounds were then used as standards to confirm the presence of 4 and 5 and their respective 13R and 13S isomers in bovine serum samples. Tetrahydroagathic acid was shown to be the only metabolite detected in serum samples taken from a suspected cattle abortion case submitted for diagnosis; and, thus, 5 could be a valuable diagnostic marker for pine needle-induced abortions.     

Development of enzyme-linked immunosorbent assays for the hepatotoxic alkaloids riddelliine and riddelliine N-oxide

Pyrrolizidine alkaloid-containing plants are widely distributed throughout the world and are particularly common in the genus Senecio. The structural types and concentrations of the alkaloids vary among plant species. In addition, within a species of plant, concentrations vary with environment and location. Many pyrrolizidine alkaloids are toxic and cause poisoning in livestock and in humans. Rapid, sensitive, and specific diagnostic techniques are needed to identify poisoned animals and to determine the particular plants and conditions under which livestock are likely to be poisoned. In this study, two competitive inhibition enzyme-linked immunosorbent assays for riddelliine, riddelliine N-oxide, and other closely related pyrrolizidine alkaloids were developed using polyclonal antibodies. One assay is class specific toward the free base forms of the pyrrolizidine alkaloids; the other assay showed cross-reactivity to both the free base and N-oxide forms of the alkaloids. The assay with the lowest limit of detection had an I(50) of 803.9 pg with a limit of detection of 47.5 pg for riddelliine. Spike and recovery studies for riddelliine in bovine blood ranged from 45 to 74%. The assay that showed cross-reactivity between the N-oxide and free base forms of the pyrrolizidine alkaloids allowed estimation of the total pyrrolizidine alkaloid content in Senecio riddellii in admixture with alfalfa. These findings suggest that these techniques will be excellent tools to diagnose poisoned animals and identify highly toxic plants.     

Glyphosate toxicity and the effects of long-term vegetation control on soil microbial communities

We assessed the direct and indirect effect of the herbicide glyphosate on soil microbial communities from ponderosa pine (Pinus ponderosa) plantations of varying site quality. Direct, toxic effects were tested using culture media and soil bioassays at glyphosate concentrations up to 100-fold greater than expected following a single field application. Indirect effects on microbial biomass, respiration, and metabolic diversity (Biolog and catabolic response profile) were compared seasonally after 9–13 years of vegetation control using repeated glyphosate applications in a replicated field study. Three pine plantations were selected to provide a range of soil characteristics associated with glyphosate binding (clay, Fe and Al oxide content) and site growing potential from the lowest to the highest in northern California. Glyphosate was toxic to bacteria and fungi from each plantation when grown in soil-free media. Culturable populations were reduced, as was the growth rate and metabolic diversity of surviving bacteria, by increasing concentrations of glyphosate. This toxicity was not expressed when glyphosate was added directly to soil, however. Microbial respiration was unchanged at expected field concentrations (5–50 μg g⁻¹), regardless of soil, and was stimulated by concentrations up to 100-fold greater. Increased microbial activity resulted from utilization of glyphosate as an available carbon substrate. Estimated N and P inputs from glyphosate were inconsequential to microbial activity. Long-term, repeated applications of glyphosate had minimal affect on seasonal microbial characteristics despite substantial changes in vegetation composition and growth. Instead, variation in microbial characteristics was a function of time of year and site quality. Community size, activity, and metabolic diversity generally were greatest in the spring and increased as site quality improved, regardless of herbicide treatment. Our findings suggest that artificial media assays are of limited relevance in predicting glyphosate toxicity to soil organisms and that field rate applications of glyphosate should have little or no affect on soil microbial communities in ponderosa pine plantations.     

Volatile and water-soluble inhibitors of nitrogen mineralization and nitrification in a ponderosa pine ecosystem

Summary Bioassay experiments were performed to test for inhibition of the processes of nitrogen mineralization and nitrification by organics in the forest floor of a ponderosa pine ecosystem. Water-extractable organics in the forest floor were tested by applying filtered extracts to the assay soil. The extract decreased nitrate production by 17.0% and decreased net mineralization by 4.1%. Inhibition by volatile organics was tested by placing vials containing forest floor or selected terpenoids of ponderosa pine in sealed jars containing the assay soil. Nitrate production was inhibited by 87.4% and 100%, and net nitrogen mineralization was inhibited by 73.3% and 67.7% in the jars with forest floor and terpenoids, respectively. Organics which are partially water-soluble and are volatile (such as terpenoids) would be very effective inhibitors of nitrogen cycling processes.     

Development of enzyme-linked immunosorbent assays for toxic larkspur (Delphinium spp.) alkaloids

Larkspur (Delphinium spp.) poisons thousands of cattle on western rangelands each year. Because poisoning does not cause specific lesions, and poisoned animals are rarely found before they die, definitively identifying poisoned animals is difficult. Additionally, toxin concentrations in larkspur plants vary with environment, plant, and location. Rapid, sensitive, and specific diagnostic techniques are needed to identify poisoned animals and to determine when and what plants are likely to poison livestock. In this study, three competitive inhibition enzyme-linked immunosorbent assays (CI-ELISA) for toxic larkspur alkaloids were developed. One assay is class-specific toward the N-(methylsuccinimido)anthranoyllycoctonine (MSAL) alkaloids, and two assays are specific for individual alkaloids. The assay with the lowest limit of detection had an I(50) of 191 pg with a limit of detection of 30.5 pg for methyllycaconitine. Spike and recovery studies using bovine blood and brain tissue ranged from 52 to 89%. These findings suggest that with additional development these techniques are likely to be excellent tools for diagnosing poisoned animals and identifying highly toxic plants.     

Impacts of acid precipitation on coniferous forest ecosystems

This paper summarizes the results from current studies in Norway. One main approach is the application of artificial acid ‘rain’ and of lime to field plots and lysimeters. Application during two growth seasons of 50 mm mo^?1 of ‘rain water’ of pH 3 to a podzol soil increased the acidity of the humus and decreased the base saturation. The reduction in base saturation was mainly due to leaching of Ca and Mg. Laboratory experiments revealed that decomposition of pine needles was not affected by any acid ‘rain’ treatment of the field plots. Liming slightly retarded the decomposition. No nitrification occurred in unlimed soils (pH 4.4-4.1). Liming increased nitrification. The soil enchytraeid (Ohgochaeta) fauna was not much affected by the acidification. Germination of spruce seeds in acidified mineral soil was negatively affected when soil pH was 4.0 or lower. Seedling establishment was even more sensitive to increasing soil acidity. Analysis of throughfall and stemflow water in southernmost Norway reveals that the total deposition of H₂SO₄ beneath spruce and pine is approximately two times the deposition in open terrain. A large part of this increase is probably due to dry deposition. Increased acidity of the rain seems to increase the leaching of cations from the tree crowns. Tree-ring analysis of spruce (Picea abies (L.) Karst.) and pine (Pinus sylvestris L.) has been based on comparisons between regions differently stressed by acid precipitation and also between sites presumed to differ in sensitivity to acidification. No effect that can be related to acid precipitation has yet been detected on diameter growth.     

Combined effects of nitrogen and elevated CO2 on soils from controlled environment studies

This paper describes the effects of elevated CO₂ and N fertilization on soils planted with ponderosa pine (Pinus ponderosa) seedlings in short-term greenhouse studies. The high degree of homogeneity in the soils used allowed sensitive evaluation of soil changes in response to treatments. Elevated CO₂ had no detectable effect upon soil N availability, but both CO₂ and N fertilization caused significant changes in soil available (NH₄F/HCl-extractable) P. Some of these changes could be accounted for by plant uptake, some were apparently due to differences in P immobilization (biotic or abiotic). N fertilization caused reductions in exchangeable K, Ca and Mg which could not be accounted for by plant uptake and were probably due to increased leaching. None of the reductions in soil available nutrients observed were of sufficient magnitude to cause nutrient deficiencies over the approximate 1-year duration of these studies.     

Among-Provenance Variability of Gas Exchange and Growth in Response to Long-Term Elevated CO2 Exposure

Genetic variability can have profound effects on the interpretation of results from elevated CO₂ studies, and future forest management decisions. Information on which varieties are best suited to future atmospheric conditions is needed to develop future forest management practices. A large-scale screening study of the effects of elevated CO₂ on 15 half-sibling sources of genetically superior ponderosa pine (Pinus ponderosa Dougl ex P. Laws.) is presented. These sources represent multiple elevations and latitudes throughout California. Among-provenance variability in the effects of elevated CO₂ on gas exchange and growth, and their correlation with geographic origin were investigated in ponderosa pine seedlings subjected to ambient or elevated CO₂ concentrations (525 μmol mol^-1 CO₂, and 700 μmol mol^-1 CO₂) for more than two years in open-top chambers. Substantial among-provenance variability in growth response to elevated CO₂ was evident, with 8 sources demonstrating no significant growth response to elevated CO₂ while 7 sources responded positively. For all sources, elevated CO₂ increased photosynthesis (ranging from 19% increase at 525 μmol mol^-1 CO₂ to 49% increase at 700 μmol mol^-1 CO₂). A modest correlation existed between geographic origin and above ground growth response to elevated CO₂.     

Trace analysis of chloramphenicol residues in eggs, milk, and meat: comparison of gas chromatography and radioimmunoassay

A radioimmunological assay (RIA) to detect chloramphenicol (CAP) residues in eggs, milk, and meat is described. For tissues and other edible products of chloramphenicol-treated animals (chickens, cows, and pigs), the limit of detection is about 200 ng/kg. Residue levels above 1 microgram/kg can easily be quantitated. When highly specific antisera produced in sheep were used, cross-reactivity was insignificant except for metabolites deviating from the parent compound in the acyl side chain only. Thiamphenicol fails to bind to the antisera; hence, it does not interfere with the assay. In the procedure described, the role of cleanup is merely to remove lipids. Thus, skim milk can be analyzed following appropriate dilution without cleanup. The results obtained by RIA were confirmed by gas chromatography with electron capture detection. The new RIA allows rapid, sensitive, and specific screening of large numbers of samples.     

Development of a real-time PCR and a sandwich ELISA for detection of potentially allergenic trace amounts of peanut (Arachis hypogaea) in processed foods

Hidden allergens in food products are, especially for peanut-allergic consumers, a serious problem because even low amounts (approximately 200 microg) of peanut can elicit allergic reactions. Undeclared peanut traces can be found in processed food products, because contaminations with peanut during production processes are frequent. To minimize the risk of such cross-contaminations, it is necessary to develop sensitive analytical methods for the detection of hidden allergens in foods. For this approach we developed two peanut-specific assays based on the detection of peanut protein by specific antibodies (sandwich ELISA) and by the detection of peanut-specific DNA (part of the coding region of Ara h 2) by a real-time PCR. Both tests did not show any cross-reactivity with 22 common food ingredients (cereals, nuts, legumes), and the limit of detection is <10 ppm peanut in processed foods. Thirty-three random samples of food products were tested for the presence of peanut to compare both assay types with each other and to evaluate the percentage of foods on the German market that are contaminated with peanut traces. We found that four products (13.3%) without peanut in the list of ingredients contained peanut protein in a range from 1 to 74 ppm peanut protein and that the results of both tests correlated well. The real-time PCR was able to detect one more positive sample than the sandwich ELISA. In conclusion, both assays are sensitive and specific tools for the detection of hidden allergens in processed foods.     

Deciduous Conifers: High N Deposition and O3 Exposure Effects on Growth and Biomass Allocation in Ponderosa Pine

Ponderosa pines (Pinus ponderosa Dougl. ex. Laws) 21 to 60 yr old were used to assess the relative importance of environmental stressors (O₃, drought) versus an enhancer (N deposition) on foliar retention, components of aboveground growth, and whole tree biomass allocation. Sites were chosen across a well-described gradient in ozone exposure (40 to 80 ppb per h, 24 h basis, 6 month growing season) and nitrogen deposition (5 to 40 kg ha^-1 yr^-1) in the San Bernardino Mountains east of Los Angeles, California. A high level of chlorotic mottle indicated high O₃ injury at sites closest to the pollution source, despite potential for the mitigating effects of N deposition. At the least polluted site, foliar biomass was evenly distributed across three of the five needle-age classes retained. At the most polluted site, 95% of the foliar biomass was found in the current year's growth. High N deposition and O₃ exposure combined to shift biomass allocation in pine to that of a deciduous tree with one overwintering needle age class. Based on whole tree harvests, root biomass was lowest at sites with the highest pollution exposure, confirming previous chamber exposure and field studies. Aboveground growth responses in the high-pollution sites were opposite to those expected for O₃ injury. Needle and lateral branch elongation growth, and measures of wood production increased with increasing proximity to the pollution source. An enhancement of these growth attributes suggested that N deposition dominated the ponderosa pine response despite high O₃ exposure.     

Characterization of pine nuts in the U.S. market, including those associated with "pine mouth", by GC-FID

Taste disturbances following consumption of pine nuts, referred to as "pine mouth", have been reported by consumers in the United States and Europe. Nuts of Pinus armandii have been associated with pine mouth, and a diagnostic index (DI) measuring the content of Δ5-unsaturated fatty acids relative to that of their fatty acid precursors has been proposed for identifying nuts from this species. A 100 m SLB-IL 111 GC column was used to improve fatty acid separations, and 45 pine nut samples were analyzed, including pine mouth-associated samples. This study examined the use of a DI for the identification of mixtures of pine nut species and showed the limitation of morphological characteristics for species identification. DI values for many commercial samples did not match those of known reference species, indicating that the majority of pine nuts collected in the U.S. market, including those associated with pine mouth, are mixtures of nuts from different Pinus species.     

Enzyme-linked immunosorbent assay for T-2 toxin metabolites in urine

A direct competitive enzyme-linked immunosorbent assay (ELISA) for determination of total T-2 toxin metabolites in urine was developed. The assay involves coating anti-3-acetyl-neosolaniol-hemisuccinate-bovine serum albumin conjugate (anti-3-Ac-NEOS-HS-BSA) antibody to the ELISA plate and using 3-Ac-NEOS-HS-peroxidase as the enzyme marker. Competitive ELISA revealed that the antibody had good cross-reactivity with acetyldiacetoxyscirpenol (Ac-DAS), T-2 tetraol tetraacetate, 3'-OH-Ac-T-2, 3-Ac-NEOS, and 3,4,15-triacetyl-12,13-epoxytrichothec-9-en-8-one (Ac-T-2-8-one), but less cross-reactivity with Ac-T-2 toxin and T-2 toxin. All metabolites of T-2 toxin in urine were converted to T-2 tetraol tetraacetate (T-2-4ol-4Ac) by acetylation of the sample extract before ELISA. To test the ELISA accuracy, a radioimmunoassay (RIA) was performed simultaneously. The linear portion of the standard curve of this direct ELISA for T-2-4ol-4Ac was 0.2-2.0 ng/mL, which was 10 times more sensitive than RIA. The minimum detection level for T-2-4ol-4Ac was 0.02 ng/mL (0.4 pg/assay) in the absence of urine sample. The overall analytical recoveries for T-2 toxin, HT-2, T-2-4ol, 3'-OH-HT-2, NEOS, and a mixture of these 5 toxins added to the urine samples in the ELISA at concentrations of 0.05 and 0.2 ng/mL were 87 and 94%, respectively.     

A method for total phosphorus determinations in nitric‐perchloric digests of pine needles and roots

Abstract

A 2:1 nitric/perchloric acid plant digest was developed and tested as a replacement of the standard Kjeldahl digest for determining phosphorus concentrations in Pinus ponderosa Laws. needles and roots. Time sequence studies using the National Institute of Standards and Technology (NIST) pine standard reference material (SRM #1575) showed increased recovery of phosphorus as digest time increased from 1.5 to 16 hours. Digest intervals greater than 16 hours did not increase phosphorus recovery; the phosphorus concentration leveled off within the NIST reference material certified range. Using P ponderosa needle and root samples, a linear regression comparison determined that two sets of nitric/perchloric digests yielded 3.4% higher phosphorus concentrations than the Kjeldahl digest. However, 99 sets of nitric/perchloric digests, each containing two NIST standard reference materials (SRM), yielded an approximately normal distribution of phosphorus concentrations, all agreeing with the Kjeldahl established concentration and within the NIST SRM certified range. The 3.4% difference in phosphorus concentrations was consistant with the observed variability among sets. In conjunction with new elemental combustion methods for determining plant nitrogen, the nitric/perchloric digest can replace a Kjeldahl digest for determining plant phosphorus while determining additional plant cations not available using a Kjeldahl digest method.     

Persistent effects of fire-induced vegetation change on energy partitioning and evapotranspiration in ponderosa pine forests

We compared energy fluxes between a site converted from ponderosa pine (Pinus ponderosa) forest to sparse grassland by a severe wildfire 10 years ago and a nearby, unburned forest. We used eddy covariance and associated instruments to measure total radiation, net radiation, albedo, and fluxes of energy into latent heat, sensible heat, and the soil. Total radiation, vapor pressure deficit, and air temperature were similar for each site. Compared to the unburned site, net radiation efficiency (net radiation/total radiation) was 30% lower and albedo 30% higher at the burned site. The magnitude of sensible and latent heats varied seasonally at both sites. Sensible heat was the major component of the energy balance in cold or dry seasons, whereas latent heat was the major component in the warm and wet season. Soil heat flux was the smallest in magnitude of the measured energy fluxes. Compared with the unburned forest, the burn-created grassland generally had lower sensible and latent heats, but greater soil heat flux for both soil cooling in winter and warming in summer. The grassland had similar maximum air temperature as the forest, and warmer surface soil temperature during the summer. Thus, the lower albedo and greater sensible heat of the forest did not produce a warmer site compared with the grassland, apparently because of the cooling effect of greater latent heat in the forest. Our results suggest only small changes in site air temperature, but larger changes in site surface soil temperature by shifts from forest to grassland caused by severe fire in northern Arizona ponderosa pine forests.     

Foliar Damage,Ion Content,and Mortality Rate of Five Common Roadside Tree Species Treated with Soil Applications of Magnesium Chloride

Sensitivity to magnesium chloride (MgCl₂) was assessed on five common roadside tree species by maintaining soil concentrations at 0-, 400-, 800-, or 1,600-ppm chloride via MgCl₂ solution over four growing seasons. Evaluations of growth, leaf retention, foliar damage, and ion concentrations were conducted. Water potentials were measured on two species. Foliar chloride and magnesium concentrations were positively correlated with foliar damage in all species. Conifers exhibited mild damage during the first growing season but moderate to severe damage during the first winter and second growing season. The two highest MgCl₂ treatments caused leaf loss, severe damage, or mortality of Douglas-fir, lodgepole, and ponderosa pines after two seasons of treatments and of limber pine after four seasons. Aspen also displayed foliar damage and crown loss but abscised damaged leaves and flushed asymptomatic leaves throughout the growing seasons. The highest treatment caused mortality of aspen in 4 years. Approximately 13,000–17,000-ppm foliar chloride was associated with severe damage in conifers but ranged from 13,000- to 33,000-ppm in fully necrotic leaves. Aspen foliage contained the highest concentrations of chloride (24,000–36,000-ppm), and limber pine leaves had the lowest (<14,200-ppm). Although MgCl₂ caused reductions in leaf water potential, aspen and ponderosa pine did not appear to be under substantial moisture stress and continued to take up ions. Mortality of common roadside tree species in 2 to 4 years can occur due to high MgCl₂ soil concentrations, and transportation officials should consider these implications in their management plans.     

Signatures of lipid assemblages in soils under continental Mediterranean forests

The analysis of free lipids in 12 soils from three representative types of Mediterranean forest has been carried out in an attempt to describe diagnostic molecules reflecting differences between the ecosystems and the intensity of the soil organic matter turnover. The study centred on the analysis by gas chromatography–mass spectrometry of the soil compounds extracted with petroleum ether from soils in central Spain, developed under monospecific formations of stone pine (Pinus pined), evergreen oak (Quercus rotundifolia) and Spanish juniper (Juniperus thurifera), the lipid extracts from their leaves also being analysed as reference material. The comparison between the distribution patterns of alkanes and fatty acids in plant lipids and the corresponding soil lipids was used to assess the extent to which the former accumulate in soil or are substituted by other biogenic or diagenetic homologues. In general, the alkane patterns showed the greatest variation in soils under oak, and the differences between lipid patterns in plant and soil were greatest in the juniper forests. As indicators of the vegetation type, the soil fatty acids had little value. Up to 60 major cyclic compounds were identified, including mainly di- and sesquiterpenes, in addition to some monoterpenes and nonterpenic naphthalenes and decalins. Of these major constituents, 33 compounds were found in soil but were not present in plant extracts, and 18 compounds were identified in plants but were not in the soils. The results suggest an arrangement of the soil samples based on the composition of the signature lipid assemblages.     

Use of the chloroplast gene ycf1 for the genetic differentiation of pine nuts obtained from consumers experiencing dysgeusia

Pine nuts are a part of traditional cooking in many parts of the world and have seen a significant increase in availability/use in the United States over the past 10 years. The U.S. Food and Drug Administration (US FDA) field offices received 411 complaints from U.S. consumers over the past three years regarding taste disturbances following the consumption of pine nuts. Using analysis of fatty acids by gas chromatography with flame ionization detection, previous reports have implicated nuts from Pinus armandii (Armand Pine) as the causative species for similar taste disturbances. This method was found to provide insufficient species resolution to link FDA consumer complaint samples to a single species of pine, particularly when samples contained species mixtures of pine nuts. Here we describe a DNA based method for differentiating pine nut samples using the ycf1 chloroplast gene. Although the exact cause of pine nut associated dysgeusia is still not known, we found that 15 of 15 samples from consumer complaints contained at least some Pinus armandii, confirming the apparent association of this species with taste disturbances.