Use ofLemna minor L. as a bioassay in allelopathy

Investigations in allelopathy often require the use of a bioassay for evaluating limited quantities of potentially active growth regulators. A bioassay procedure was developed usingL. minor grown in 1.5-ml aliquots of nutrient medium with and without allelochemicals in wells of 24-well tissue culture cluster dishes with loose-fitting lids. Tests using six replications per treatment with several flavonoid compounds and derivatives of coumarin, benzoic acid, and cinnamic acid demonstrated that the bioassay was capable of measuring inhibition at levels of compound ranging from 50 to 1000 mol. Strongly inhibitory treatments were visible after 1 or 2 days. After 7 days of growth, frond number, growth rate, and dry weight were used to evaluate effects. The bioassay system is relatively simple, very sensitive, reproducible, and can be used for testing small amounts and dilute concentrations of unknowns which have been separated by chromatography.Supported under USDA Specific Cooperative Agreement No. 58-32U4-3-507. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the U.S. Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.     

Sensitivity ofLemna bioassay interacts with stock-culture period

The effect of stock-culture period on the sensitivity of theLemna bioassay to four different phenolic substances was evaluated in this study. The sensitivity of the bioassay interacted with the stock-culture period of either 11, 14, or 18 days. After a seven-day testp-hydroxybenzoic acid (HBA), vanillic acid (VA),trans-cinnamic acid (CA), and ferulic acid (FA) with a concentration of 0.05 mM significantly reduced the number of fronds and the dry weight compared with the untreated control when the stock-culture period had been exactly 14 days. The sensitivity after the shorter (11 days) or longer (18 days) stock-culture period was reduced, and the differences in the dry weight to the untreated control were not significant after a stock culture period of 18 days. The two higher concentrations (0.1 and 0.25 mM) showed stronger inhibition. A comparison of the inhibition at 0.05 mM revealed that the stock-culture period affected the relative toxicity of the four phenolic substances. Since the pH increased in the stock-culture flasks during the 18-day period from 6.25 to 7.9, we hypothesize that differences in theLemna assay can be at least partly attributed to a pH effect, possibly in combination with a relative nutrient deficiency. We recommend a standardization of the stock-culture period to 14 days to improve the sensitivity of theLemna bioassay.     

Effects of juglone on growth,photosynthesis, and respiration

The impacts of juglone on plant growth and several other physiological functions were evaluated in this study. Juglone inhibitedLemna minor growth, chlorophyll content, and net photosynthesis at treatments between 10 and 40M. Soybean leaf disks vacuum infiltrated with as little as 10M juglone had reduced photosynthesis. Oxygen evolution by chloroplasts isolated fromPisum sativum was inhibited by juglone with an I₅₀ of 2M. Micromolar treatments of juglone stimulated oxygen uptake in mitochondria isolated fromGlycine max. These data suggest perturbations of chloroplast and mitochondrial functions may contribute to plant growth reductions observed in juglone-mediated allelopathy.This study was supported by National Science Foundation grant STI-8902066.     

Effects of three phenolic compounds onLemna gibba G3

Lemna gibba L. G3, (duckweed) was used as a bioassay organism to test the allelochemical effects of salicylic acid (SA), ferulic acid (FA), and umbelliferone (UM). Growth rate (K), dry weight (DW) and total chlorophyll (CHL) production were measured after seven days of growth. The bioassay procedure used 50 ml of E medium with and without sucrose in 125-ml Erlenmeyer flasks plus the selected concentration of allelochemical. At concentrations of 50 M and greater, SA caused inhibition of K and DW production inL. gibba G3, while the threshold for CHL reduction was 20 M. FA inhibited the DW and CHL production at 100 M when the compound was auto-claved in E medium containing sucrose. Treatments of UM were least toxic with an inhibition threshold of 500 M for K and DW production in medium without sucrose. UM did not reduce CHL production until 750 M. In some cases, different thresholds of inhibition were observed depending on the presence or absence of sucrose and tartaric acid in the medium, and whether or not the chemicals were autoclaved with the medium.This research was presented by the senior author as partial fulfillment for the Master of Arts degree, Biology Department, Hood College, Frederick, Maryland. Mention of a trade name or proprietary product does not constitute a guarantee or warranty by the USDA and does not imply its approval to the exclusion of other products that may be suitable.     

Identification and Synthesis of the Sex Pheromone of the Passionvine Mealybug, Planococcus minor (Maskell)

The sex pheromone of the mealybug, Planococcus minor was isolated by fractionation of crude pheromone extract obtained by aeration of virgin females. The pheromone was identified as the irregular terpenoid, 2-isopropyl-5-methyl-2,4-hexadienyl acetate, by mass spectrometry, microchemical tests, and ¹H NMR spectroscopy. The stereochemistry of the pheromone was assigned as (E) by comparison with synthetic standards of known geometry. The compound was highly attractive to males in laboratory bioassays, whereas the (Z)-isomer appeared to antagonize attraction.     

Effects of three phenolic acids on chlorophyll content and growth of soybean and grain sorghum seedlings

Experiments were designed to test the hypothesis that interference with chlorophyll metabolism may be one mechanism of inhibition of plant growth in allelopathic interactions. Effects of ferulic,p-coumaric, and vanillic acids on soybean and grain sorghum growth and chlorophyll content were quantified and compared after seedlings were treated with these compounds in a nutrient culture. Following a 6-day treatment cycle, dry weights of soybean seedlings were reduced by both 10^–3 M and 5 × 10^–4 M treatments of ferulic,p-coumaric and vanillic acids. Soybean weight reductions in each case were paralleled by a significant reduction in the concentration (g Chl/mg dry wt) of chlorophylls a and b and total chlorophyll in the unifoliate leaves. Sorghum seedling growth was also reduced by each of the compounds at the 5 × 10^–4 M level, but leaf chlorophyll concentration was not below that of control plants.     

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.     

Distribution and antifeedant associations of sesquiterpene lactones in cultivated sunflower (Helianthus annuus L.) on western corn rootworm (Diabrotica virgifera virgifera LeConte)

Seven antifeedant sesquiterpene lactones (STLs), 4,5-dihydroniveusin A, argophyllin B, argophyllin A, 15-hydroxy-3-dehydrodesoxytifruticin, niveusin B, 1,2-anhydridoniveusin A, and an unidentified epoxide, in cultivated sunflower (Helianthus annuus L.) have been quantified by a highperformance thin-layer chromatography and UV-reflectance scanning densitometry analysis. Age-related expression of STL content in sunflower reveals a heretofore undescribed pattern in which nonpolar STLs such as 15-hydroxy-3-dehydrodesoxytifruticin predominate up to an age of three weeks, but are subsequently displaced by polar STLs, especially argophyllin A, through later foliar stages and anthesis. This leaf pattern of STL ontogeny is maintained in three widely differentH. annuus cultivars (Giant Gray Stripe, Royal Hybrid 2141, Hybrid 7111), which in turn had similar total contents of STLs. Antifeedant activity for western corn rootworm was positively correlated with STL content, particularly with argophyllin A and its isomer argophyllin B, in respective tissue extracts. Enhanced amounts of highly antifeedant argophyllins, especially in newly grown leaf and floral tissues yielding sunflower progeny, strongly suggest that these epoxy-STLs are a chemical defense against insect herbivory.     

Influence of cnicin,a sesquiterpene lactone ofCentaurea maculosa (Asteraceae), on specialist and generalist insect herbivores

The sesquiterpene lactone cnicin was extracted fromCentaurea maculosa andCentaurea vallesiaca. We examined its effects on the ovipositional response and larval development of generalist and specialist insect herbivores associated withC. maculosa. For the oviposition trials, three plant species (C. maculosa, Achillea millefolium, andCichorium intybus), half of which were sprayed with 3% of cnicin, were exposed to the specialist mothsStenodes straminea, Agapeta zoegana, andPterolonche inspersa in field cages. All three species significantly preferredC. maculosa to other plants andP. inspersa significantly preferred cnicin-sprayed plants to untreated plants for oviposition. Tested over all species, cnicin significantly increased the number of eggs laid on a given plant. A larval diet test examined the toxicity of cnicin for larvae of the generalist noctuid mothSpodoptera littoralis. Cnicin concentrations of 3% and 6% were lethal and 1% and 0.5% seriously inhibited growth and development. The larvae of theC. maculosa specialistStenodes straminea survived at 6% cnicin, but none of the pupae hatched.Agapeta zoegana was able to survive at 1% and 3% cnicin. Both specialists had difficulties with the artificial diet, but weight increase and survival was not further reduced when cnicin was present compared with on the control diet. In conclusion, cnicin influenced host recognition by the specialist species, and larvae of the generalist did not survive on natural levels of cnicin. Growth and survival of the specialist were not influenced by cnicin but were considerably hampered on artificial diet.     

Allelopathic effect of phenolic acids from humic solutions on two spruce mycorrhizal fungi:Cenococcum graniforme andLaccaria laccata

The aim of this investigation was to determine the impact ofp-hydroxyacetophenone,p-hydroxybenzoic acid, catechol, and protocatechuic acid on respiration of two spruce mycorrhizal fungi:Laccaria laccata andCenococcum graniforme. These phenols are produced byVaccinium myrtillus,Athyrium filixfemina, andPicea abies, predominant species of spruce forests in the Alps, and they are also present in humic solutions at 10^–10 M or 10^–5 M. Respiration of the two fungi was inhibited by the four phenolic acids, even at concentrations ranging from 10^–5 M to 10^–7 M. These data show phenolic acids from humic solutions have biological activity at extremely low concentrations, suggesting a contribution ofV. myrtillus, A. filixfemina, andP. abies to allelopathic inhibition of mycorrhizal fungi.     

Differences in attraction to semiochemicals present in sympatric pine shoot beetles,Tomicus minor andT. piniperda

The chemical ecology of host- and mate-finding in the pine shoot beetles,Tomicus minor andT. piniperda, was studied in southern Sweden. Beetles were collected in the field from defined attack phases on Scots pine. Using gas chromatography-mass spectroscopy, a number of oxygen-containing monoterpenes, e.g., 3-carene-10-ol, myrtenol,trans-verbenol, and verbenone, were identified from hindgut extracts of both sexes of both species. Compared toT. minor,T. piniperda contained additional compounds and in larger amounts. The amounts were highest in both species at the time when the beetles had bored into contact with the resin-producing xylem-phloem tissue. The synthesis of (1S,6R)-3-carene-10-ol by photooxidatipn of (+)-(1S,6R)-3-carene is described. In comparative electroantennogram (EAG) measurements on males and females of both species, the most active of the tested compounds wastrans-verbenol. Laboratory bioassays of walking beetles showed thatT. piniperda was attracted to uninfestèd pine logs.T. minor was more strongly attracted to pine logs infested with females than to uninfested pine logs, indicating a female-produced aggregation pheromone. Field tests confirmed thatT. piniperda was strongly attracted to pine logs. The attraction ofT. minor to logs was significant only when logs were combined with racemictrans-verbenol and (1S,6R)-3-carene-10-ol.T. minor was also attracted to a combination of these monoterpene alcohols alone. We suggest that host and mate location inT. piniperda is achieved by means of a kairomone composed of host monoterpenes, whileT. minor utilizes a primitive pheromone synergized by host odors. Evolution of host colonization strategies of the two beetles are discussed.Coleoptera: Scolytidae.This study was made within the project Odour signals for control of pest insects.     

Allelopathic effects of phenolic mixtures on respiration of two spruce mycorrhizal fungi

Investigations were conducted to determine whether the allelopathic effect (inhibition of oxygen consumption) of mull- and mor-type humic solutions on ectomycorrhizal fungi was due to the solutions' phenolic contents. In the first experiment, two concentrations (10^–3 M and 10^–7 M) of binary equimolar phenolic mixtures were tested on the oxygen consumption ofLaccaria laccata andCenococcum graniforme. The high concentrations of most of the mixtures induced an increase of fungal respiration, whereas the 10^–7 M treatments all reduced the rate of respiration. In the second experiment, the effects on respiration of the phenolic mixture reproducing mull- and mortype humic solutions were compared with the effects of natural humic solutions. The resulting data suggest that allelopathic effect of humic solutions can at least in part be attributed to their phenolic contents.     

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

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

Effects of agingmortierella mycelium on production of arachidonic and eicosapentaenoic acids

Arachidonic acid and eicosapentaenoic acid (EPA) are important intermediates of eicosanoid metabolism and are presently the subject of extensive nutritional and medical research. The effects of mycelial aging on production of these fatty acids were investigated as part of a research program directed toward examining the feasibility of economically producing these products by fungal fermentation. Arachidonic acid content ofM. alpina ATCC 32222 increased from 4.1–8.3% to 13–16% during aging while lipid content of mycelium increased from 14–18% to 33–45%. Maximum lipid content produced in biomass during storage declined as harvesting time was increased from 3 to 6 days while maximum arachidonic acid content in lipid increased. Maximum lipid and arachidonic acid was produced during aging at pH 8, whereas arachidonic acid content of lipids was highest in mycelium aged at pH 6. EPA content ofM. elongata NRRL 5513 biomass increased during aging, reaching a maximum after 22–28 days. When the pH of the culture prior to harvesting was adjusted in the range of pH 4–9, pH values for development of maximum EPA in biomass and in lipids during storage were found to be 6 and 7, respectively. Temperature of aging had little effect on arachidonic acid or EPA content.     

Phenolic acids affect photosynthesis and protein synthesis by isolated leaf cells of velvet-leaf

The effects ofp-coumaric, ferulic, chlorogenic, and vanillic acids on photosynthesis and protein synthesis by isolated leaf cells of velvetleaf (Abutilon theophrasti Medik) were investigated. Photosynthesis and protein synthesis were measured in cell suspensions by the incorporation of¹⁴CO₂ and [¹⁴C]leucine, respectively. None of the tested phenolic acids except vanillic reduced photosynthesis by more than 50% at the highest concentration and 30 min of incubation. At 100M concentrations and 60-min incubation periods,p-coumaric, ferulic, chlorogenic, and vanillic acids inhibited photosynthesis by 33, 37, 57, and 65%, respectively. Ferulic acid was the most inhibitory to protein synthesis and reduced the incorporation of [¹⁴C]leucine by 50% at about 1.0M after 60 min of incubation. At the highest concentrations tested in this study, vanillic and ferulic acids were inhibitory to photosynthesis and protein synthesis, respectively, whereas chlorogenic andp-coumaric acids did not inhibit either physiological process. The maximum inhibition of protein synthesis by chlorogenic acid was 19% and by vanillic acid was 28% at 100M concentrations. Chlorogenic, vanillic, andp-cou-maric acids at 0.1M caused increased protein synthesis over the untreated control. Overall, photosynthesis was more sensitive than protein synthesis to the four phenolic acids tested.Florida Agricultural Experiment Station Journal Series No. 9228.     

Effects of mineral nitrogen andBradyrhizobium inoculation on growth and iron nutrition of groundnut

Experiments were conducted in a glasshouse to determine the effects of the mineral N supplied as ammonium nitrate andBradyrhizobium inoculation on the growth and iron nutrition of nodulating and non-nodulating groundnut (Arachis hypogaea L.) lines. In a sterilized sand-vermiculite medium supplied with N-free nutrient solution (pH 7.0), inoculation of nodulating groundnut withBradyrhizobium strain NC 43.3 enhanced dry matter production and O-phenanthroline extractable iron and N contents of the plants. The supply of mineral N at a rate of 100 mg N L^–1 (as NH₄NO₃) through deionized water (pH 8.5) induced iron chlorosis symptoms in the nodulating groundnuts grown in Vertisols, but these symptoms were not observed at higher N levels (200–400 mg N L^–1). The induced chlorosis was only partially corrected by inoculation withBradyrhizobium strains NC 92 and NC 43.3. The iron deficiency chlorosis was, however, corrected by application of higher rates of ammonium nitrate.Submitted as JA No. 942 of the International Crops Research Institute for the Semi-Arid Tropics (ICRISAT)     

Effects of mixtures of phenolic acids on phosphorus uptake by cucumber seedlings

To determine how individual phenolic acids in a mixture might affect phosphorus (P) uptake, 15-day-old cucumber seedlings grown in solution culture were treated with ferulic, vanillic,p-coumaric, or equimolar mixtures of these phenolic acids. Phenolic acid and P uptake were determined by solution depletion. The joint action of the mixtures of these phenolic acids on P uptake was primarily additive. Thus, as the number of phenolic acids increased in the mixture, the concentrations of the individual phenolic acids in the mixture required to bring about a given response declined. Seedling uptake of individual phenolic acids from solution mixtures of phenolic acids was reduced when compared to the uptake of phenolic acids from single phenolic acid solutions. The magnitude of the reduction varied with phenolic acid and concentration. The dose required for 50% inhibition of P uptake was approximately two to three times higher for vanillic acid (6.73 mM) than for ferulic (2.27 mM) andp-coumaric acids (3.00 mM) when dose was based on the initial treatment concentrations. The dose required for 50% inhibition of P uptake was not significantly different for the three phenolic acids (42 ± 5 mol/g root fresh weight) when dose was based on phenolic acid uptake. Potential reasons for these differences are discussed.Paper No. 12527 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, North Carolina 27695-7643. The use of trade names in this publication does not imply endorsement by the North Carolina Agricultural Research Service of products named, nor criticism of similar ones not mentioned. This research was partially supported by the US-Spain Joint Committee for Scientific and Technological Cooperation project CCA-8309/166.     

Effects of some compounds isolated fromCelaenodendron mexicanum standl (euphorbiaceae) on seeds and phytopathogenic fungi

The known compounds friedelin, maytensifolin B, ginkgetin, bilobetin, and amentoflavone as well as the new triterpene 3-hydroxyfriedelan-16-one were isolated fromCelaenodendron mexicanum (Euphorbiaceae), an endemic tree of the Pacific Ocean coast of Mexico. The compounds' structures were established on the basis of spectral analysis. The biological effects of aqueous leachates, a CHCl₃-MeOH extract and the isolated compounds of leaves and twigs were evaluated on the radicle growth of two plants,Amaranthus leucocarpus andEchinochloa crusgalli, and on the radial growth of three phytopathogenic fungi,Fusarium sp.,Helminthosporium sp., andAlternaria sp. The organic extracts of both leaves and twigs inhibitedAmaranthus and stimulatedEchinochloa radicle growth. On the contrary, friedelin and maytensifolin B stimulatedAmaranthus and inhibitedEchinochloa. The target fungi showed a different response to each treatment, from inhibition to stimulation.Taken in part from the M.Sc. thesis of P. Castañeda.On sabbatical leave from Departamento de Preparatoria Agrícola Universidad Autónoma de Chapingo, México.     

Effects of selected substrate forms on the synthesis of structured lipids by two immobilized lipases

Two immobilized lipases, IM 60 from Rhizomucor miehei and SP 435 from Candida antarctica, were used to synthesize structured lipids (SL). Tricaprin and trilinolein were interesterified to produce SL that contained one linoleic acid per triacylglycerol molecule (SL1) and SL with two linoleic acids (SL2). SL1 and SL2 were separated by silver nitrate thin-layer chromatography according to their unsaturation, and the fatty acid at the sn-2 position was determined after pancreatic lipasecatalyzed hydrolysis of SL1 and SL2. With IM 60, 57.7 mol% capric acid and 42.3 mol% linoleic acid were found at the sn-2 position of SL1, while 43.3 mol% capric acid and 56.7 mol% linoleic acid were at the sn-2 position of SL2. The fatty acid at the sn-2 position of SL1 with SP 435 as biocatalyst was 43.6 mol% capric acid and 56.4 mol% linoleic acid, while SL2 contained 56.6 mol% capric acid and 43.4 mol% linoleic acid. Different structural forms of the capric acid-containing substrate (triacylglycerol vs. ethyl ester) and different chainlengths of triacylglycerol were selected to study the substrate selectivity of lipases. Results indicated that SP 435 had some degree of preference for the triacylglycerol form (tricaprin), and IM 60 produced SL more rapidly and reached steady state faster with tricaprin as substrate than with capric acid ethyl ester. For chainlength selectivity, mol% of synthesized SL from tricaprin + trilinolein and tristearin + trilinolein were compared. SP 435 exhibited no apparent preference for either tricaprin or tristearin. However, IM 60 showed a more rapid reaction with tricaprin than with tristearin.     

Allelopathic Effects of Volatile Cineoles on Two Weedy Plant Species

The volatile monoterpene analogs, 1,4-cineole and 1,8-cineole, have been identified as components of many plant essential oils, but relatively little is known about their biological activities. We compared the effects of 1,4- and 1,8-cineole on two weedy plant species by monitoring germination, mitosis, root and shoot growth, chlorophyll content, and photosynthetic efficiency. 1,4-Cineole severely inhibited growth of roots and shoots, causing cork-screw shaped morphological distortion, whereas 1,8-cineole caused a decrease in root growth and germination rates. Chlorophyll fluorescence data (yield and F _v / F _m) indicated that 1,4-cineole caused significantly higher stress (P 0.001) to photosynthesis when compared to controls. Mitotic index data showed that 1,8-cineole severely decreased (P 0.001) all stages of mitosis when compared with controls, while 1,4-cineole only caused a decrease in the prophase stage (P 0.05). Although superficially similar in structure, these two cineoles appear to have different modes of action.