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.     

Relationships betweenDendroctonus micans Kug. (Coleoptera: Scolytidae) survival and development and biochemical changes in Norway Spruce,Picea abies (L.) Karst., phloem caused by mechanical wounding

The bark of Norway spruce trees,Picea abies (L.) Karst., was wounded to produce areas of bark with differing biochemical characteristics. Adults and larvae ofDendroctonus micans Kug. were inserted into these trees at points around the wounds. Larval survival was higher, larval dry weights were higher, and adults were more likely to oviposit at sites around the wound that had higher phloem moisture contents. Larvae showed higher survival and dry weights in phloem with low terpene contents. Adults oviposited in phloem with lower stilbene glucoside contents. Phloem moisture differed at sites around the wound compared with that at a control site away from the wound, and the nitrogen content of entire bark cores was higher above wounds than at control sites away from the wounds. Phloem moisture correlated with the nitrogen content of entire bark. Phloem terpene contents correlated with each other, as did the stilbene contents. Associations between the host exploitation pattern ofD. micans and the biochemistry of the host phloem are discussed in relation to preselection by adults of sites suitable for larval development.     

Monoterpene metabolism in female mountain pine beetles,Dendroctonus ponderosae Hopkins,attacking ponderosa pine

Abdominal volatiles of female mountain pine beetles,Dendroctonus ponderosae Hopkins, fed in ponderosa pine,Pinus ponderosa Dougl. ex Laws, and in lodgepole pine,P. contorta var.latifolia Engelmann, were analyzed by gas chromatography and coupled gas chromatography-mass spectrometry and were found to comprise host oleoresin components and beetle-produced alliylic alcohols, aldehydes, and ketones derived from host monoterpenes. Neitherexo- andendo-brevicomin nor frontalin were detected. Three metabolic pathways are proposed to account for the distribution of beetle-produced monoterpene alcohols. The first pathway involves hydroxylation of monoterpene substrates on allylic methyl groups which areE to a methylene or vinyl group. This oxidation pathway is indiscriminate with respect to substrate and probably functions to detoxify monoterpenes. A second pathway, which hydroxylates theendo-cyclic methyleneE to a vinyl methyl group of bicyclic monoterpenes to give almost exclusively thetrans alcohol, is hypothesized to be involved in pheromone production. A third detoxification pathway involves anti-Markovnikov addition of water to theexo-cyclic double bond of -phellandrene to give predominantlytrans-2-p-menthen-7-ol.Coleoptera: Scolytidae.Research supported by the Natural Sciences and Engineering Research Council of Canada: Operating Grant A3881 and A3706; Strategic Grant G1039; and Science Council of British Columbia Grant 33 (RC-4).     

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.     

Monoterpene concentrations in fresh,senescent, and decaying foliage of singleleaf pinyon (Pinus monophylla Torr. & Frem.: Pinaceae) from the western Great Basin

Senescent foliage from pines is potentially a large contributor to the total monoterpene content of the litter layer, and the availability of these compounds as phytotoxins may result from release of these compounds into the vapor phase. In order to determine the fate of several monoterpene hydrocarbons in the natural environment, we examined their concentrations in fresh, senescent, and decaying needles from 32 single-leaf pinyon pine (Pinus monophylla Torr. & Frem.: Pinaceae) trees growing at two different locations. Total monoterpene content was highest in the fresh needles (mean=5.6 ± 2.2 mg/g extracted air dry weight), but also remained relatively high in senescent needles (mean=3.6 ±1.8 mg/g extracted air dry weight), either still attached to the tree or forming the freshest layer of understory litter. Decaying needles within a dark decomposing layer of litter material 5–20 cm from the surface were found to contain much lower amounts of total monoterpenes (average: =0.12 ±0.06 mg/g extracted air dry weight). Further investigation of the fate of these compounds in the pinyon understory is required to determine if these hydrocarbons are indeed exerting phytotoxic characteristics.     

Structural Requirements of Jasmonates and Mimics for Nicotine Induction inNicotiana sylvestris

Jasmonic acid (JA) is strongly implicated in the long-distance signal transduction cascade increasing nicotine synthesis in the roots of plants after leaf wounding. In order to explore the structural requirements of the inducing signal, we examined jasmonates, mimics, and a biosynthetic precursor for nicotine-inducing activity (NIA). We examine the importance of the keto group on the five-membered ring and the double bond in then-pentenyl chain by comparing the NIA of methyl jasmonate (MJ) with that of cucurbic acid, 1,3-dithiolane-MJ, 1,3-dioxolane-MJ, methyl dihydrojasmonate (DHMJ), 1,3-dioxolane-DHMJ, 1-oxo-indan-4-carboxylic acid ILE-methyl ester, and 1-hydroxyl-indan-4-carboxylic acid ILE-methyl ester. We found that: 1,3-dioxolane MJ, cucurbic acid, and 1,3-dioxolane DHMJ were less active than MJ and that the isoleucine (ILE) conjugates of 1-oxo- and l-hydroxyindanon-4-carboxylic acid had the same NIA as MJ. The activities of these indanon amino acid conjugates may be due to the structural similarity of their keto or hydroxyl groups on the five-membered ring to MJ or to the keto-enolized MJ. These results support the hypothesis that the enolization of the keto group during or prior to its interaction with the putative JA receptor is required for activity. We explore the importance of the esterification of the carboxyl functional group by comparing the NIAs of cucurbic acid and cucurbic acid methyl ester, l-oxo-indan-4-carboxylic acid, 1-oxo-indan-4-carboxylic acid methyl ester, and l-oxo-indan-4-carboxylic acid ILE-methyl ester. In all cases, the esters were more active than the free acids. We compared the NIA of MJ of different epimeric composition (8% and 20% 3R,7S-MJ); 12-oxophytodienoic acid (12-oxo-PDA) methyl ester, an important precursor of JA; and coronatine (a well-known phytotoxin and putative structural mimic of 12-oxo-PDA).We found that: (1) the epimeric composition of MJ did not affect its NIA; (2) 12-oxo-PDA methyl ester had lower NIA than MJ; and (3) coronatine significantly inhibited plant growth but did not increase nicotine biosynthesis. In summary, JA, rather than its biosynthetic precursor, 12-oxo-PDA, is likely the endogenous signal inNicotiana sylvestris, and the keto functional group on the five-membered ring and the double bond in then-pentenyl side chain are crucial components of JA for NIA.     

Piperidine Alkaloids in Nitrogen Fertilized Pinus ponderosa

We fertilized individual, pole-size ponderosa pine trees at two low-quality sites and pine saplings at a relatively high-quality site, with ammonium nitrate. Six to 12 months later, we measured total %N and 2,6-disubstituted piperidine alkaloids in the foliage. The N additions raised foliar %N above deficiency levels (i.e., from 1.0–1.1% to 1.4–1.6%) at the low-quality sites, but did not elevate foliar %N in saplings at the higher quality site, where it was already (1.9%) well above critical levels. In control trees with foliar N below a threshold of 1.1%, we detected no more than trace levels of alkaloids, indicating that alkaloid production is highly constrained by N deficiency. The N additions increased mean concentrations of the predominant alkaloid, pinidine, at all three sites. Mean total alkaloid concentrations for fertilized trees at the two low-quality sites were 12 and 155 g/g dry wt higher than controls (relative increases of 12× and 4.5×, respectively). For saplings at the high-quality site, the mean total increased by 584 g/g dry wt (1.6×) with the N additions. Allocation of foliar N to alkaloids was highest in fertilized saplings (0.81%) compared to control saplings (0.53%). These findings demonstrate that foliar alkaloid concentrations can be increased by nitrogen fertilization of forest trees growing on both low- and high-quality sites. Fertilizing for the purpose of inhibiting potential herbivores may be more successful at higher quality sites where alkaloid levels are enhanced relative to food quality (foliar %N).     

Effect of a Rugulosin-producing Endophyte in Picea glauca on Choristoneura fumiferana

Disease-free second instar Choristoneura fumiferana (eastern spruce budworm) were placed on trees infected with a rugulosin-producing needle endophyte in two experiments. They were allowed to grow to sixth instar when survivors were collected. First, by using 3-yr old trees, a comparison was made of budworm growth on infected and uninfected trees. A second experiment used 4-yr old trees to study the effect of rugulosin content in the needle on growth. This permitted an examination of a dose response in relation to growth, and allowed us to eliminate the potential for differences in environment or foliar chemistry affecting the results. At sixth instar, budworms feeding on infected trees that contained rugulosin were smaller than those on uninfected trees. At needle concentrations above the dietary low observed effect level of rugulosin for C. fumerana >0.5 μg g⁻¹, a dose response was seen. For the first time, this demonstrates an inverse effect in outdoor nursery experiments between budworm weight and rugulosin concentration.     

Variation of enantiomeric composition of α-pinene in norway spruce,Picea abies,and its influence on production of verbenol isomers byIps typographus in the field

The enantiomeric composition of -pinene in individual Norway spruce trees [Picea abies (L.) Karst.] was determined on a chiral GC column after stereoselective hydroboration-oxidation followed by a reaction with isopropyl isocyanate to form the carbamate derivative. The enantiomeric composition varied considerably between trees of different genetic origin. There was a strong correlation between the chirality of -pinene in host spruce trees and thecis/trans ratio of verbenols found in the hindguts of the bark beetleIps typographus (L.) infesting the trees.Coleoptera; Scolytidae.Parts of this work have been presented at the 14th International Symposium on the Chemistry of Natural Products in Poznan, Poland, July 9–14, 1984.     

Antioxidant effects of flavonoids ofAnthriscus sylvestris in lard

Ethanol/water (7∶3) extracts of the plant speciesAnthriscus sylvestris possess antioxidant activity. Separation and identification of antioxidant components by thin-layer and column chromatography and spectral analysis demonstrated that quercetin and apigenin appeared to be the main flavonoid species inAnthriscus sylvestris. Rutin was one of the major quercetin glycosides. Structures of the isolated compounds were determined by infrared and¹H nuclear magnetic resonance spectroscopies. Ethanolic extract (70%) ofA. sylvestris showed concentration-dependent, strong antioxidant activity as determined by the Schaal Oven test of lard at 60°C. Rancimat analysis at 100°C showed that the antioxidant activity of 70% ethanolic extract ofA. sylvestris was superior to quercetin, apigenin, or a tocopherol mixture.     

Effects of bilberry (Vaccinium myrtillus L.) litter on seed germination and early seedling growth of four boreal tree species

Laboratory and greenhouse bioassays were used to test for inhibitory effects of senescent and decomposed leaves and aqueous extract from bilberry (Vaccinium myrtillus L.) against seed germination and seedling growth of aspen (Populus tremula L.), birch (Betula pendula Roth.), Scots pine (Pinus sylvestris L.), and Norway spruce [Picea abies (L.) Karst.]. Aqueous extracts from bilberry leaves were inhibitory to aspen seed germination and seedling growth and also induced root damage and growth abnormalities. Addition of activated carbon removed the inhibitory effects of extracts. Senescent leaves reduced pine and spruce seed germination, but rinsing of seeds reversed this inhibition. Senescent leaves were more inhibitory than decomposed leaf litter, suggesting that the inhibitory compounds in bilberry leaves are relatively soluble and released at early stages during decomposition. Spruce was generally less negatively affected by litter and aqueous extracts than the other tested species. This study indicates that chemical effects of bilberry litter have the potential to inhibit tree seedling recruitment, but these effects were not consistently strong. Phytotoxicity is unlikely to be of critical importance in determining success for spruce seedling establishment.     

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).     

Relationships Between Phenolics and Soil Microorganisms in Spruce Forests: Significance for Natural Regeneration

Plant-produced phenolic compounds inhibit soil microbial activity and are suspected to be involved in the failure of natural regeneration of French subalpine Norway spruce (Picea abies) forests. This work evaluated relationships between phenolic compounds and soil microorganisms in humus collected in spruce forest during winter and summer seasons. After having identified and quantified different phenolics from humus samples, we incubated another set of humus samples with 10 times the concentration of naturally occurring phenolics, and effects on the density of microorganisms were compared to humus with no addition of phenolic compounds. Furthermore, in order to follow the degradation of phenolic compounds by microbes, the concentration of phenolics in the incubated humus samples was determined by HPLC after three and six days of incubation. The results indicate that humus microorganisms use phenolics as a carbon source, since almost all phenolic compounds had disappeared after six days of incubation. Addition of phenolic compounds to the humus samples also affected the soil microbial populations so that bacteria were inhibited in the humus collected both in the winter and in the summer. However, there were differences between the experiments. Fungi and cellulose hydrolyzers were stimulated in the winter humus experiment, while fungi were unaffected and cellulose hydrolyzers were inhibited in the summer experiment. The addition of phenolic compounds stimulated ammonifiers in the summer experiment. We anticipate that the role of soil microorganisms in the problem of natural regeneration failure is probably more important than previously thought.     

Multivariate study of moose browsing in relation to phenol pattern in pine needles

Extracts of needles from Scots pine,Pinus sylvestris L., grown on fertile (A) or poor (B) soil, and from lodgepole pine,P. contorta Doug. (C), have been analyzed with HPLC and UV detection. By application of multivariate analysis, the phenol pattern was shown to vary between species and between habitats. Within groups A and B, moose food choice was highly correlated to phenol pattern. Concentrations of most of the analyzed compounds were internally correlated. Information related to pine growth rate could also be extracted from the phenolic pattern for A, B, and C. Moose food choice was not correlated to pine growth rate.     

Measurement of monoterpene hydrocarbon levels in vapor phase surrounding single-leaf pinyon (Pinus monophylla Torr. & Frem.: Pinaceae) understory litter

A headspace air-sampling experiment was performed in the laboratory to determine the identity and concentrations of monoterpene hydrocarbons that could be attained in the vapor phase surrounding single-leaf pinyon pine (Pinus monophylla Torr. & Frem.) understory litter using controlled air collection conditions at a simulated field temperature of 37.8°C. The total monoterpene hydrocarbon content in 21 sequential samples of air collected from a sealed glass carboy packed with 1.44 kg of single-leaf pinyon litter equivalent to a bulk density of 0.15 g/cm³ averaged 3.56 ± 1.04 mg/liter. The monoterpenes-pinene and camphene were present in the vapor phase at the highest concentrations, averaging 2.40±0.64 and 0.68±0.22 mg/ liter, respectively. Myrcene,-pinene, 3-carene,-phellandrene, and-terpinene were all present at average concentrations below 0.30 mg/liter. The first two traps of the sequential air samples yielded the highest concentrations for the monoterpene hydrocarbons; however, the average total levels were relatively stable throughout the remaining 19 traps. Therefore, the data indicate that these hydrocarbons volatilize from the source pinyon litter and maintain an equilibrium in the vapor phase. Although this analysis was conducted using an artificial system, combined with results from our previous studies, mounting evidence indicates that monoterpene hydrocarbons present in the vapor phase of the single-leaf pinyon understory may be toxic to a variety of native plant species and thus further implicates allelopathy as a significant contributor to the observed patterning of associated vegetation in these forests.     

Fatty acids of the seeds from pine species of the Ponderosa-Banksiana and Halepensis sections. The peculiar taxonomic position of Pinus pinaster

The fatty acid compositions of pine seed oils were determined from 11 species of the Banksiana subsection and three species of the Ponderosa subsection. All were collected in North America (United States, Mexico, and Cuba). These analyses also included the seed oils from the unique European species of the Ponderosa-Banksiana section (Banksiana subsection), Pinus pinaster, and from three pine species of the Halepensis section, which are related to the Banksiana subsection. Emphasis was placed on their Δ5-olefinic acid content and profile. Principal-component analysis of fatty acid compositions showed that all North American species constituted a fairly homogeneous group. However, P. jeffreyi was slightly eccentric, and P. pinaster, a west-Mediterranean species, was completely isolated from the North American group. Other species from the Banksiana and Ponderosa subsections could not be distinguished on the basis of their seed oil fatty acid compositions. With respect to Δ5-olefinic acids, the North American species (except for P. jeffreyi) had 5,9-18:2, 5,9,12-18:3, 5,11-20:2, and 5,11,14-20:3 acid concentrations in the ranges 1.9 to 3.2, 17.7 to 22.9, 0.2 to 0.4, and 2.0 to 3.5%, respectively (sum, 22.7–28.5%). Levels of corresponding acids in P. pinaster were 0.9, 7.9, 0.9, and 7.0%, respectively (sum, 16.7%). Other differences were observed for linoleic acid (42.6 to 48.6% vs. 52.2%) and α-linolenic acid (0.3 to 0.6% vs. 1.4%). Pinus pinaster was close to species of the Halepensis section (5,9-18:2, 0.5 to 1.0%; 5,9,12-18:3, 3.1 to 4.4%; 5,11-20:2, 0.4 to 0.5%; 5,11,14-20:3, 3.6 to 5.4%; sum, 8.6–11.1%), which were clearly separated from the Ponderosa-Banksiana section. Among all pines analyzed, P. pinaster presented the highest level of sciadonic (5,11,14-20:3) acid, a component that has three ethylenic bonds in common with arachidonic and eicosapentaenoic acids.     

Effects of octadecanoid metabolites and inhibitors on induced nicotine accumulation inNicotiana sylvestris

We examined the effects of inhibitors of the octadecanoid pathway (n-propyl gallate, acetosalicylic acid, salicylhydroxamic acid, methyl salicylate, and antipyrine) on wound- and jasmonate-induced nicotine accumulation and compared the nicotine-inducing ability of exogeneous additions of linolenic acid (18:3) and its methyl ester, linoleic acid (18:2), abscisic acid, traumatic acid, and methyl dihydrojasmonate to the nicotine-inducing ability of exogenous additions of methyl jasmonate (MJ). The first four of these inhibitors significantly reduced wound-induced nicotine accumulation when applied in a lanolin paste to wounded tissues immediately after wounding at concentrations of 89–90µg/plant. When methyl salicylate and propyl gallate were mixed individually with MJ, neither inhibited MJ-induced nicotine synthesis, which suggests that the inhibitors block jasmonate synthesis or release from stored pools and not its effects. Linolenic acid or its methyl ester applied to undamaged plants or damaged plants (to either damaged or undamaged leaves) or to the roots of hydroponically growing plants did not induce nicotine accumulation or increase nicotine accumulation above levels found in damaged plants. Similarly, traumatic acid, linoleic acid, and abscisic acid did not induce nicotine accumulations. Methyl dihydrojasmonate, which is biosynthetically derived from linoleic acid, had 12–56% of the nicotine-inducing acitivity of MJ when added to the roots of hydroponically grown plants. The signal transduction pathway mediating wound-induced nicotine production therefore shares many features of the pathway eliciting wound-induced proteinase inhibitor production but differs in not being regulated at the lipase step in jasmonic acid production and not being responsive to abscisic acid.     

Comparing the Variation of Needle and Wood Terpenoids in Scots Pine Provenances

We determined variation in both the concentration and composition of terpenoids in needles and wood within nine Scots pine (Pinus sylvestris) provenances. Seedlings of different provenances representing a 1200-km N–S transect from Estonia to northern Finland were cultivated in Suonenjoki nursery field, central Finland, for seven years. Growth of seedlings and the number of vertical resin ducts in wood were also determined. -Pinene and 3-carene were the major monoterpenes both in the needles and wood. The total monoterpene concentration was about five times higher in the needles than in the wood. A strong positive correlation was found between proportional quantities of several terpenes of the needles and wood, particularly for 3-carene, sabinene, and terpinolene. The needles contained both labdane-type and tricyclic resin acids, whereas the wood contained only tricyclic ones. The wood had a four times higher tricyclic resin acid concentration than the needles. The highest total monoterpene concentration in the needles and in the wood occurred in the most northern Muonio provenance and in the most southern Saaremaa provenance plants, respectively. The amount of high 3-carene genotype trees decreased among the northern provenances. The wood of the most northern Muonio provenance showed the lowest total resin acid concentration, but provenance did not affect total tricyclic resin acids in the needles. Korpilahti provenance trees from central Finland had the best growth in height. In addition, Korpilahti and Ruokolahti provenance trees showed largest radial growth of stem and smallest number of vertical resin ducts. The results suggest that especially the proportional quantity of 3-carene in the needles could be used in estimating the amount of this compound in the wood and vice versa.     

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.     

Chemical response of Picea glehnii seed-epiphytic Penicillium species to Pythium vexans under in vitro competitive conditions for mycelial growth

The potential protection of Picea glehnii seedlings from damping-off by seed-epiphytic Penicillium species was investigated. We studied the chemical response of seed-epiphytic Penicillium species (Pen. cyaneum, Pen. damascenum, and Pen. implicatum) to Pythium vexans, a damping-off fungus, in vitro. Penicillium species were cultured singly or cocultured with Pyt. vexans for 14 or 18 d, and mycelial growth, pH of culture filtrate, antifungal activity of the culture filtrate against Pyt. vexans, and the amount of antifungal compound produced by each Penicillium species, were examined. The filtrate of both the single culture of Penicillium and the coculture of Penicillium and Pyt. vexans showed antifungal activity against Pyt. vexans. In a coculture with Pyt. vexans, Pen. cyaneum produced an antifungal compound (patulin) as in the single culture. Pen. damascenum cocultured with Pyt. vexans produced an antifungal compound (citrinin), as it did in the single culture and in larger amounts on day 10. Pen. implicatum produced two antifungal compounds, frequentin and palitantin, and the ratio of frequentin (with higher antifungal activity than palitantin) to palitantin was higher in the coculture with Pyt. vexans than in the single culture. Our results indicate that these Penicillium species have the ability to produce antifungal compounds and to keep antifungal activity under competitive condition with Pyt. vexans. The chemical response of these Penicillium species to Pyt. vexans may contribute to protect P. glehnii seedlings from damage by Pyt. vexans.