Screening of Ophiostoma Species for Removal of Eucalyptus Extractives

Abstract

Ophiostoma species have been demonstrated to metabolize wood extractives and be useful to the pulp and paper industry. In order to have new isolates for the Asian market, Eucalyptus camaldulensis logs were harvested from forest sites in central Taiwan and 28 strains of the Ophiostoma genus were isolated from them. These strains were subsequently inoculated onto Eucalyptus wood chips to evaluate their effects on weight losses of wood and the amounts of acetone extractives degraded. At the same time, Gas Chromatography-Mass Spectroscopy (GC-MS) analysis was conducted and by using calibration curves and a database of GC-MS mass spectra, changes in lipophilic compounds were analyzed. Fatty acids, hydrocarbons, sterol compounds, sterol esters, and triglycerides were significantly reduced after two weeks’ inoculation by the fungal strains. The results show that six of the strains were capable of reducing the lipophilic fractions by more than 60% in a two-week treatment. DNA of the most effective strains were analyzed and found to be a variant of Ophiostoma querci.     

Antiherbivore Chemistry of Eucalyptus--Cues and Deterrents for Marsupial Folivores

Formylated phloroglucinol compounds (FPCs) are the single most important factor determining the amount of foliage that marsupial folivores eat from individual Eucalyptus trees. Folivores need to recognize which trees contain FPCs if they are to avoid them and forage efficiently, they are challenged by great diversity in the types and quantities of FPCs present, even within eucalypt species. We investigated the relationship between FPCs and terpenoids in species with both simple and complex FPC profiles and found strong positive correlations between terpenes generally, and several monoterpenes in particular, and FPCs. Terpene cues also indicated qualitative differences in trees' FPC profiles. We describe significant qualitative and quantitative variation in FPCs in several species that are important food sources for marsupial folivores. New discoveries include the fact that macrocarpals occur as two major, distinct groups and several new dimeric acylphloroglucinols from Eucalyptus strzeleckii. These patterns add to the chemical complexity of the foraging environment for folivores.     

Influence of odor from wood-decaying fungi on host selection behavior of deathwatch beetle,Xestobium rufovillosum

Adult females of Xestobium rufovillosum de Geer demonstrated anemotactic orientation when exposed to an odor plume containing volatiles generated by wood-decaying fungi (Coriolus versicolor, Donkioporia expansa) and decayed oak wood (Quercus petraea, Quercus robur). They did not orient towards undecayed oak wood, beech (Fagus sylvatica), or pine wood (Pinus sylvestris). Although all insects tested showed anemotactic orientation, responses were nonlinear with respect to insect age. Adult females more readily oriented upwind when they were between 10 and 16 days old. Oviposition choice bioassays showed that ovipositing females would preferentially oviposit on extract-treated cellulose paper discs that had been treated with various strains of the wood-decaying fungus, Donkioporia expansa. HPLC-fractionated mycelial extracts were attractive to ovipositing deathwatch beetles, whereas HPLC-fractionated fungal broth extracts were repellent to ovipositing females. The implications of these results are discussed in the context of timber pest management in historically important buildings.     

Phenolic Metabolites in Leaves of the Invasive Shrub, Lonicera maackii, and Their Potential Phytotoxic and Anti-Herbivore Effects

Lonicera maackii is an invasive shrub in North America for which allelopathic effects toward other plants or herbivores have been suspected. We characterized the major phenolic metabolites present in methanol extracts of L. maackii leaves. In addition, we examined the effects of methanol–water extracts of L. maackii leaves on seed germination of a target plant species and on feeding preference and growth rate of a generalist insect herbivore. A total of 13 individual major and minor compounds were detected in crude leaf extracts by high-performance liquid chromatography coupled to electronspray ionization-tandem mass spectrometry (ESI-MS/MS). Extracts were dominated by two major flavones, apigenin and luteolin, and their glucoside derivatives, apigenin-7-glucoside and luteolin-7-glucoside. Quantities of these compounds, along with chlorogenic acid, varied between two sampling points. Leaf extracts that contained these compounds were inhibitory to seed germination of Arabidopsis thaliana. In addition, treatment of artificial diet with leaf extracts deterred feeding of the generalist herbivore, Spodoptera exigua, in choice experiments but had no effect on growth rate in short-term no-choice bioassays. Purified apigenin tended to deter feeding by S. exigua and inhibited seed germination of A. thaliana. We conclude that leaves of L. maackii contain phenolic compounds, including apigenin and chlorogenic acid, capable of having biological effects on other plants and insects.     

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.     

Effect of Wood Welding Process on Chemical Constituents of Australian Eucalyptus

Thermochemical changes occurring during wood welding were investigated in Eucalyptus saligna and Eucalyptus pilularis. Unwelded reference wood and material from welded interface were compared via Py-GC/MS, thermogravimetric analyses, X-ray photoelectron spectroscopy, and attenuated total reflection Fourier transform infrared spectroscopy to explain differences in mechanical properties of welded wood between species. It appeared that the species originally containing more condensed substructures also provided stronger joints. The condensation index after welding allowed validating that the adhesive properties of lignin are more accessible in such species. The presence of more carbonyl functions, attributed to carbohydrate solicitation during welding process, potentially made lignin less accessible. Changes following welding were evidenced by the contribution of extractive compounds, fatty acid chains, and terpenoids, possibly reacting with hydroxyl groups and leading to adhesive properties by chemical linkage through new covalent bonds formation. Results corroborate the better mechanical properties of E. saligna, providing stronger joints possibly due to more accessible adhesive properties by esterification between lignin and fatty acid.     

Soy protein combined with copper and boron compounds for providing effective wood preservation

Chromated copper arsenate (CCA), an arsenicbased wood preservative, is toxic to human health and the environment. Although CCA is stable in seasoned wood, there are potential dangers during CCA manufacture, lumber treatment, and waste disposal. This research was conducted to study the effectiveness of soy products to replace toxic chromium and arsenic compounds in wood preservative formulations. Three soy product (Arpro 2100, HM 90, and Supro 760) were used as fixative agents in preservative solutions containing anhydrous CuSO₄ and Na₂B₄O₇·10H₂O. The decay resistance of treated wood blocks was measured by a soil-block culture method. Despite the large molecular sizes of copper-protein and copper-boron-protein complexes, southern pine sapwood was treatable with these preservative formulations. Wood samples treated with >6 kg°m⁻³ CuSO₄ and 7.5 kg·m⁻³ soy product, and subsequently leached for 3 d and exposed to the decay fungus Gloeophyllum trabeum (Fr.) Mur., sustained only 0.5% weight loss over 12 wk. Wood samples needed 40 kg·m⁻³ CuSO₄ and 50 kg·m⁻³ soy protein to resist the copper-tolerant decay fungus Postia placenta (Fr.) M. Lars. & Lomb. These results suggest that soy-based wood preservatives can prevent wood products from fungal attack and can replace CCA.     

Evolution of Extractive Composition During Thermal Treatment of Jack Pine

Abstract

The thermal treatment of wood has many benefits such as better dimensional stability and attractive dark color and does not use toxic chemicals. The resistance against biological decay can be improved when wood is not in contact with ground. On the other hand, after thermal transformation, wood becomes more fragile. The changes of the wood properties are related to the modification of the wood composition. During the thermal treatment, the evaporation of the moisture content is not the only event. Volatile extractives are evacuated from the wood, while new products and by-products of different chemical reactions appear. The comparison of the extracts obtained from untreated and treated wood can help to identify thermo-chemical reactions, taking place during the heat treatment. This article presents the analysis by Gas Chromatography–Mass Spectroscopy (GC-MS), High Performance Liquid Chromatography (HPLC), and Thin Layer Chromatography (TLC) of polar and non-polar extracts of untreated and heat-treated North American Jack pine (Pinus banksiana). The study of the impact of maximum heat treatment temperature on the composition of the Jack pine extracts showed that the major part of extractives leaves the wood under 200°C whereas most of the new products appear only above 200°C. While the extractives of the untreated Jack pine are dominated by non-polar components, the thermo-transformation seems to generate mainly polar compounds. However, presence of water vapor increases the portion of polar extractives in wood. Interestingly, an important decrease of concentration of phenolic compounds (such as pinosylvin, pinosylvin monomethyl ether, and pinobanksin) in Jack pine wood was observed between 160–200°C. On the other hand, 4-hydroxy-methylfurfural and vanillin have been identified as compounds generated by the heat treatment above 200°C. The identification of other by-products will be presented in a later paper.     

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.     

Controlled release of biocides in solid wood. III. Preparation and characterization of surfactant‐free nanoparticles

Polymeric nanoparticles containing the fungicides tebuconazole and chlorothalonil were prepared by a simple, surfactant‐free method and found to have significantly smaller median particle diameters and more stable aqueous suspensions than their surfactant‐stabilized counterparts. These more stable suspensions were delivered into southern yellow pine and birch wood with greater efficiency than the equivalent surfactant‐stabilized nanoparticle suspensions. We found that the suspensions protected the treated wood against fungal attack by Gloeophyllum trabeum, a common brown rot wood decay fungus, and Trametes versicolor, a common white rot wood decay fungus, at low tebuconazole and chlorothalonil contents in the wood. Southern pine lost 5% or less of its mass after 55 days of exposure to G. trabeum when the tebuconazole or chlorothalonil content in the wood was only 0.4 kg/m³, while a tebuconazole or chlorothalonil content of 0.8 kg/m³ in birch wood was sufficient to bring its mass loss to less than 5% after 55 days of exposure to T. versicolor. © 2002 Wiley Periodicals, Inc. J Appl Polym Sci 86: 615–621, 2002     

Chemical Defenses of Freshwater Macrophytes Against Crayfish Herbivory

We measured feeding preferences of the crayfish Procambarus clarkii for fresh tissue from four species of freshwater macrophytes (Habenaria repens, Saururus cernuus, Ceratophyllum demersum and Typha angustifolia). We then determined the role of plant chemical defenses in generating these preferences by incorporating crude aqueous and organic extracts from each species into palatable foods and comparing feeding on these foods to feeding on control foods lacking these extracts. Tissue toughness, dry mass and ash-free mass per volume, and percentages of carbon, nitrogen, and phenolics were also measured for each of the four macrophytes. Although it had a low nutritional value, Ceratophyllum was the preferred food when it was offered as fresh tissue; it did not produce a chemically deterrent extract. The lipophilic crude extract from Typha significantly deterred crayfish feeding, but this highly nutritious plant was preferred when offered in an agar-based diet lacking structural defenses. Habenaria and Saururus were low preference foods that did not appear to be structurally defended; each species contained both lipophilic and water-soluble extracts that significantly deterred feeding. Fractionation of the lipophilic crude extract from Saururus indicated the presence of at least three deterrent compounds. From the orchid Habenaria, we isolated and identified a novel bis-p-hydroxybenzyl-2-alkyl-2-hydroxysuccinoate metabolite, habenariol, that appeared to explain most of the feeding deterrent activity present in the lipophilic extract of this species. The concentration of the metabolite in frozen collections of this plant doubled if we allowed the material to thaw before placing it in extraction solvents.     

HPLC identification of allelopathic compounds fromLantana camara

Aqueous extracts ofLantana camara L. leaves inhibited ryegrass (Lolium multiflorum Lam.) germination and seedling growth. Phytotoxic compounds were fractionated from crude aqueous extracts and fractions were evaluated for their phytotoxicity. Inhibition was most pronounced with the alkaline and acid hydrolysates. Plant inhibition by the crude extract reflected a complex interaction of numerous individual components of diverse chemical compositions and potencies. Presumptive identification of the individual components was accomplished with high-performance liquid chromatography (HPLC). Thirteen phenolic compounds were identified, and most of these compounds were phytotoxic to ryegrass seedlings. Radicle elongation was more sensitive to the toxins than shoot elongation.Florida Agricultural Experiment Station Journal Series No. 7906.     

Does the odor from sponges of the genus Ircinia protect them from fish predators?

Caribbean sponges of the genus Ircinia contain high concentrations of linear furanosesterterpene tetronic acids (FTAs) and produce and exude low-molecular-weight volatile compounds (e.g., dimethyl sulfide, methyl isocyanide, methyl isothiocyanate) that give these sponges their characteristic unpleasant garlic odor. It has recently been suggested that FTAs are unlikely to function as antipredatory chemical defenses, and this function may instead be attributed to bioactive volatiles. We tested crude organic extracts and purified fractions isolated from Ircinia campana, I. felix, and I. strobilina at naturally occurring concentrations in laboratory and field feeding assays to determine their palatability to generalist fish predators. We also used a qualitative technique to test the crude volatile fraction from I. felix and I. strobilina and dimethylsulfide in laboratory feeding assays. Crude organic extracts of all three species deterred feeding of fishes in both aquarium and field experiments. Bioassay-directed fractionation resulted in the isolation of the FTA fraction as the sole active fraction of the nonvolatile crude extract for each species, and further assays of subfractions suggested that feeding deterrent activity is shared by the FTAs. FTAs deterred fish feeding in aquarium assays at concentrations as low as 0.5 mg/ml (fraction B, variabilin), while the natural concentrations of combined FTA fractions were >5.0 mg/ml for all three species. In contrast, natural mixtures of volatiles transferred from sponge tissue to food pellets and pure dimethylsulfide incorporated into food pellets were readily eaten by fish in aquarium assays. Although FTAs may play other ecological roles in Ircinia spp., these compounds are effective as defenses against potential predatory fishes. Volatile compounds may serve other defensive functions (e.g., antimicrobial, antifouling) but do not appear to provide a defense against fish predators.     

Using wood extracts to determine the feeding preferences of the western drywood termite,Incisitermes minor (Hagen)

Nymphs of the western drywood termite,Incisitermes minor (Hagen), were tested for their feeding preferences on wood and extracts of 11 tree species. The amount of wood consumed was inversely proportional to its specific gravity, but methanol extracts of least preferred woods were also least preferred when termites were confined to paper treated with extracts. In choice tests, only paper treated with extract of wood from which the colony developed and untreated and methanol-treated controls were significantly fed upon. Ponderosa pine extract applied to Douglas fir and sugar pine significantly decreased the amount of wood consumed. Resistance to termite feeding appeared to depend on the presence of repellent chemicals in the wood.     

Hydroxamic Acid Content and Toxicity of Rye at Selected Growth Stages

Rye (Secale cereale L.) is an important cover crop that provides many benefits to cropping systems including weed and pest suppression resulting from allelopathic substances. Hydroxamic acids have been identified as allelopathic compounds in rye. This research was conducted to improve the methodology for quantifying hydroxamic acids and to determine the relationship between hydroxamic acid content and phytotoxicity of extracts of rye root and shoot tissue harvested at selected growth stages. Detection limits for an LC/MS-MS method for analysis of hydroxamic acids from crude aqueous extracts were better than have been reported previously. (2R)-2-β-d-Glucopyranosyloxy-4-hydroxy-(2H)-1,4-benzoxazin-3(4H)-one (DIBOA-G), 2,4-dihydroxy-(2H)-1,4-benzoxazin-3(4H)-one (DIBOA), benzoxazolin-2(3H)-one (BOA), and the methoxy-substituted form of these compounds, (2R)-2-β-d-glucopyranosyloxy-4-hydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one (DIMBOA glucose), 2,4-hydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one (DIMBOA), and 6-methoxy-benzoxazolin-2(3H)-one (MBOA), were all detected in rye tissue. DIBOA and BOA were prevalent in shoot tissue, whereas the methoxy-substituted compounds, DIMBOA glucose and MBOA, were prevalent in root tissue. Total hydroxamic acid concentration in rye tissue generally declined with age. Aqueous crude extracts of rye shoot tissue were more toxic than extracts of root tissue to lettuce (Lactuca sativa L.) and tomato (Lycopersicon esculentum Mill.) root length. Extracts of rye seedlings (Feekes growth stage 2) were most phytotoxic, but there was no pattern to the phytotoxicity of extracts of rye sampled at growth stages 4 to 10.5.4, and no correlation of hydroxamic acid content and phytotoxicity (I₅₀ values). Analysis of dose–response model slope coefficients indicated a lack of parallelism among models for rye extracts from different growth stages, suggesting that phytotoxicity may be attributed to compounds with different modes of action at different stages. Hydroxamic acids may account for the phytoxicity of extracts derived from rye at early growth stages, but other compounds are probably responsible in later growth stages.     

Comparative Phloem Chemistry of Manchurian (<Emphasis Type="Italic">Fraxinus mandshurica</Emphasis>) and Two North American Ash Species (<Emphasis Type="Italic">Fraxinus americana and Fraxinus pennsylvanica</Emphasis>)

Recent studies have investigated interspecific variation in resistance of ash (Fraxinus spp.) to the exotic wood-boring beetle, emerald ash borer (EAB, Agrilus planipennis). Manchurian ash (Fraxinus mandshurica) is an Asian species that has coevolved with EAB. It experiences little EAB-induced mortality compared to North American ashes. Host phloem chemistry, both constitutive and induced, might partly explain this interspecific variation in resistance. We analyzed the constitutive phloem chemistry of three ash species: Manchurian ash and North American white (Fraxinus americana) and green (Fraxinus pennsylvanica) ash. Analysis of the crude phloem extracts revealed the presence of an array of phenolic compounds including hydroxycoumarins, a monolignol, lignans, phenylethanoids, and secoiridoids. Both qualitative and quantitative differences were observed among the three ash species. Hydroxycoumarins and the phenylethanoids, calceloariosides A and B, were present only in the phloem of Manchurian ash and might represent a mechanism of resistance against EAB.     

Comparison of Antifungal and Antioxidant Activities of <Emphasis Type="Italic">Acacia mangium</Emphasis> and <Emphasis Type="Italic">A. auriculiformis</Emphasis> Heartwood Extracts

The effect of heartwood extracts from Acacia mangium (heartrot-susceptible) and A. auriculiformis (heartrot-resistant) was examined on the growth of wood rotting fungi with in vitro assays. A. auriculiformis heartwood extracts had higher antifungal activity than A. mangium. The compounds 3,4,7,8-tetrahydroxyflavanone and teracacidin (the most abundant flavonoids in both species) showed antifungal activity. A. auriculiformis contained higher levels of these flavonoids (3.5- and 43-fold higher, respectively) than A. mangium. This suggests that higher levels of these compounds may contribute to heartrot resistance. Furthermore, both flavonoids had strong 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity and laccase inhibition. This suggests that the antifungal mechanism of these compounds may involve inhibition of fungal growth by quenching of free radicals produced by the extracellular fungal enzyme laccase.     

Rapid Induction of Multiple Terpenoid Groups by Ponderosa Pine in Response to Bark Beetle-Associated Fungi

Ponderosa pine (Pinus ponderosa) is a major and widely distributed component of conifer biomes in western North America and provides substantial ecological and economic benefits. This tree is exposed to several tree-killing bark beetle-microbial complexes, including the mountain pine beetle (Dendroctonus ponderosae) and the phytopathogenic fungus Grosmannia clavigera that it vectors, which are among the most important. Induced responses play a crucial role in conifer defenses, yet these have not been reported in ponderosa pine. We compared concentrations of terpenes and a phenylpropanoid, two phytochemical classes with strong effects against bark beetles and their symbionts, in constitutive phloem tissue and in tissue following mechanical wounding or simulated D. ponderosae attack (mechanical wounding plus inoculation with G. clavigera). We also tested whether potential induced responses were localized or systemic. Ponderosa pines showed pronounced induced defenses to inoculation, increasing their total phloem concentrations of monoterpenes 22.3-fold, sesquiterpenes 56.7-fold, and diterpenes 34.8-fold within 17 days. In contrast, responses to mechanical wounding alone were only 5.2, 11.3, and 7.7-fold, respectively. Likewise, the phenylpropanoid estragole (4-allyanisole) rose to 19.1-fold constitutive levels after simulated attack but only 4.4-fold after mechanical wounding. Overall, we found no evidence of systemic induction after 17 days, which spans most of this herbivore’s narrow peak attack period, as significant quantitative and compositional changes within and between terpenoid groups were localized to the wound site. Implications to the less frequent exploitation of ponderosa than lodgepole pine by D. ponderosae, and potential advantages of rapid localized over long-term systemic responses in this system, are discussed.     

Foveal glands,source of sex pheromone production in the ixodid tickDermacentor andersoni stiles

The foveal glands of the Rocky Mountain wood tick,Dermacentor andersoni Stiles, are the sex pheromone glands from which the sex pheromone is released via the foveae dorsales. The sex pheromone, 2,6-dichlorophenol, was recovered from extracts of these glands by GLC. Other evidence of the role of these glands in sex pheromone production is described. A³⁶Cl-labelled volatile compound (or compounds) was (were) collected from partially engorged femaleD. andersoni fed in³⁶Cl-labelled hosts, but no labelled compounds were collected when the foveae dorsales were blocked. X-ray analysis revealed unusual concentrations of chlorine in the foveal glands compared to other tissues. Autoradiography also revealed significant accumulations of radiochlorine in the vicinity of these glands.Presumably, the foveal glands of the American dog tick,Dermacentor variabilis (Say), are the sex pheromone glands of that species also, since a³⁶Cl-labelled volatile was collected from female ticks fed on a³⁶Cl-labelled host. However, attempts to recover 2,6-dichlorophenol from gland extracts or volatile emissions fromD. variabilis were unsuccessful.Supported by grants AI 10,986 and AI 10,987 from the National Institute of Allergy and Infectious Diseases, U.S. Public Health Service, U.S. Department of Health, Education and Welfare, Bethesda, Maryland, 20014.     

Exogenous jasmonates simulate insect wounding in tomato plants (Lycopersicon esculentum) in the laboratory and field

Wounding increases the levels and activities of several defense-related proteins in the foliage of the tomato plant,Lycopersicon esculentum Mill. Evidence indicates that two of these responses, the systemic increases in polyphenol oxidase and proteinase inhibitors, are regulated by an octadecanoid-based signalling pathway which includes the wound hormone, jasmonic acid. It is not known whether other responses to wounding are also regulated by this same signalling pathway. In this paper, we show that application of jasmonates (jasmonic acid or its volatile derivative, methyl jasmonate) in low concentrations to foliage of young tomato plants induced, in a dose-dependent manner, the same protein responses-polyphenol oxidase, proteinase inhibitors, lipoxygenase, and peroxidase-as doesHelicoverpa zea Boddie feeding. Application of jasmonic acid to a single leaflet of four-leaf tomato plants induced these four proteins in a spatial pattern nearly identical to that produced by localized feeding ofH. zea. Exogenous jasmonic acid also decreased suitability of foliage for the beet armyworm,Spodoptera exigua Hubner in the laboratory. Based on these results, we conducted an experiment to measure the effects of jasmonic acid spray under field conditions. We provide the first evidence that jasmonic acid spray on field plants induces production of chemical defenses above the levels found in unsprayed controls. Exogenous jasmonic acid sprayed on plants in agricultural plots increased levels of polyphenol oxidase and proteinase inhibitors. Because application of jasmonic acid induces these defensive compounds at low concentrations in a manner similar to natural wounding, it may prove to be a useful tool for stimulating plant resistance to insects in the field.