Effects of Monoterpenoids, Acting Alone or in Pairs, on Seed Germination and Subsequent Seedling Growth

We compared the potential allelopathic activity of 47 monoterpe- noids of different chemical groups, by estimating their effect on seed germination and subsequent growth of Lactuca sativa seedlings. Apart from individual compounds, eleven pairs at different proportions were also tested. As a group, the hydrocarbons, except for (+)-3-carene, were the least inhibitory. Of the oxygenated compounds, the least inhibitory were the acetates; whenever the free hydroxyl group of an alcohol turned into a carboxyl group, the activity of the resulting ester was markedly lower (against both germination and seedling growth). Twenty-four compounds were extremely active against seedling growth (inhibiting it by more than 85%), but only five against seed germination. The compounds that were most active against both processes belonged to the groups of ketones and alcohols; they were terpinen-4-ol, dihydrocarvone, and two carvone stereoisomers. We used a model to investigate whether compounds acted independently when applied in pairs. The combined effect varied. In half of the cases, it followed the pattern expected under the assumption of independence; in the rest, either synergistic or antagonistic interactions were found in both germination and elongation. However, even in cases of synergistic interactions, the level of inhibition was not comparable to that of a single extremely active compound, unless such a compound already participated in the combination. The specific structural factors that operate and determine the activity of monoterpenoids still remain rather obscure. The same holds true for the combined effect; its character cannot in general be predicted on the basis of individual compounds acting alone.     

Dynamics of macronutrients during the first stages of litter decomposition from forest species in a temperate area (Galicia,NW Spain)

The decomposition and dynamics of nutrient elements was studied for leaves and twigs from Quercus robur, Pinus radiata and Eucalyptus nitens in soils developed from granodiorite, slate and limestone in Galicia, NW Spain. Two 1-ha plots were selected for each material-vegetation combination, making a total of 18 plots. Litter decomposition and nutrient dynamics during the first six months were investigated using litterbags. Mass loss was higher for leaves than twigs and not significantly different for different tree species. Mass loss correlated significantly with carbon loss (r = 0.96 for leaves, r = 0.90 for twigs). As a general trend, nutrient release from leaves and twigs was greater for the broadleaved species (eucalyptus and oak). K and S were rapidly released from all litters, while Ca and Mg showed the highest tendency to be immobilised. N and P behave similarly, with a final balance of net release. Soil parent material did not significantly influence decomposition or nutrient dynamics.     

Allelopathy of oats. I. Assessment of allelopathic potential of extract of oat shoots and identification of an allelochemical

The allelopathic potential of oat (Avena sativa L.) extracts was investigated under laboratory conditions. The ethyl ether-, acetone-, and water-soluble fractions obtained from the extract of oat shoots inhibited the germination and growth of roots and hypocotyls of lettuce (Lactuca sativa L.). The inhibitory activity of the water-soluble fraction was maximum, followed by that of ethyl ether-soluble and acetone-soluble fraction. An active principle of the water-soluble fraction was isolated and its structure was determined by spectral data asl-tryptophan.l-Tryptophan inhibited the growth of hypocotyls and roots of lettuce seedlings at concentrations greater than 0.03 and 0.1 mM, respectively. These results suggested thatl-tryptophan may be an allelochemical which affects the growth or germination of different plant species.     

Rumex obtusifolius L: Release of allelochemical agents and their influence on small-scale spatial distribution of meadow species

DecomposingRumex obtusifolius L. leaves and their extracts were most toxic for germination and root growth of meadow speciesLolium perenne, Trifolium repens, Poa pratensis, andDactylis glomerata after seven days' decomposition, although the toxicity level was, in some cases, still high after 21 days. The hypothesis thatR. obtusifolius exerts allelopathic control over meadow species is supported by small-scale distribution of meadow species in the neighborhood ofR. obtusifolius plants. The area affected and the intensity of the effect both increase with the size of the individualR. obtusifolius plant.     

Selective Phytotoxic Activity of Withanolides from Iochroma australe to Crop and Weed Species

Phytotoxic effects of Iochroma australe extract and 4,7,20-oxowithanolides were examined in Petri dish bioassays at 10, 100, and 1000 ppm. The extract and the major constituent (17S,20R,22R)-4,7,20-trihydroxy-1oxowitha-2,5,24-trienolide (2) reduced growth of the radicle of the weeds Sorghum halepense (Monoct.) and Chenopodium album (Dicot.). Neither the extract nor withanolides had significant effect on germination or radicle length of the commercial crop species Lactuca sativa. From our experimental data we conclude that the withanolides tested here are natural products with selective herbicidal activity against weed species.     

Biomimetic measurement of allelochemical dynamics in the rhizosphere

Polydimethylsiloxane (PDMS) materials were used to quantify levels of the photosynthesis inhibitor sorgoleone in the undisturbed rhizosphere of sorghum plants. The materials used included stir bars coated with PDMS (stir bar sorptive extraction), technical grade optical fiber coated with a thin film of PDMS (matrix-solid phase microextraction), and PDMS tubing. PDMS tubing retained the most sorgoleone. As analyzed by high performance liquid chromatography, amounts of sorgoleone retained on the PDMS materials increased with time. Other materials tested (polyurethane foam plugs, C18 and Tenax disks, and resin capsules) proved less suitable, as they were subject to sometimes extensive penetration by fine root hairs. These results demonstrate the potential for PDMS-based materials to monitor the release of allelochemicals in the undisturbed rhizosphere of allelopathic plants. Unlike extraction procedures that recover all available compounds present in the soil, PDMS functions in a manner more analogous to plant roots in sorbing compounds from soil solution or root exudates. Information on chemical dynamics in the rhizosphere is crucial for evaluating specific hypotheses of allelopathic effects, understanding allelopathic mechanisms, and assessing the importance of allelopathic processes in plant communities.     

Comparative analysis of allelopathic effects produced by four forestry species during decomposition process in their soils in Galicia (NW Spain)

The development of toxicity produced by vegetable litter of four forest species (Quercus robur L.,Pinus radiata D.Don.,Eucalyptus globulus Labill, andAcacia melanoxylon R.Br.) was studied during the decomposition process in each of the soils where the species were found. The toxicity of the extracts was measured by the effects produced on germination and growth ofLactuca saliva L. var. Great Lakes seeds. The phenolic composition of the leaves of the four species was also studied using high-performance liquid chromatographic analysis (HPLC). It was verified that toxicity was clearly reflected in the first stages of leaf decomposition inE. globulus andA. melanoxylon, due to phytotoxic compounds liberated by their litter. At the end of half a year of decomposition, inhibition due to the vegetable material was not observed, but the soils associated with these two species appeared to be responsible for the toxic effects. On the other hand, the phenolic profiles are quite different among the four species, and greater complexity in the two toxic species (E. globulus andA. melanoxylon) was observed.     

Effects of fertility on biomass,phytotoxicity, and allelochemical content of cereal rye

Studies were conducted to evaluate biomass production, tissue phytotoxicity, and allelochemical content of rye (Secale cereale L.) shoots grown in three fertility regimes (low, medium or high) in the greenhouse. Wheeler rye or a polyculture of rye and hairy vetch (Vicia villosa Roth) grown with high fertility produced the greatest biomass (78.7 and 82.7 g), with the lowest root-to-shoot ratio (0.22 and 0.43) produced in the high-fertility treatments. The polyculture treatment grown with low fertility had a greater proportion of hairy vetch (18%) than when grown with high fertility (6%). Rye shoot residue phototoxicity was affected by fertility regime. Radicle elongation of cress (Lepidium sativum L.) and barnyardgrass [Enchinochloa crus-galli (L.) Beauv. I was inhibited by rye shoot residues in a modified Parker bioassay. Rye shoot residue grown with low fertility was less inhibitory to cress radicle elongation than rye residue from the medium- or high-fertility regimes. Extracts of dried rye shoots grown with high fertility were less inhibitory than extracts from other fertility regimes. The concentrations of ether extracts of rye causing 50% inhibition (I₅₀) of cress radicle elongation were between 125 and 276 µg/ml for greenhouse-grown and 60 and 138 µg/ml for the field-grown rye shoots. The major phytotoxic compounds in the rye shoot extracts were identified as DIBOA and BOA. The concentration of DIBOA in the greenhouse-grown rye shoots ranged between 128 and 423 µg/g while BOA concentration ranged between 2.5 and 31 µg/g. DIBOA and BOA levels were lowest in rye shoots grown with high fertility. Correlations between rye shoot biomass, DIBOA and BOA concentration, and cress barnyardgrass radicle length were significant.     

Light-mediated allelochemical effects of naturally occurring polyacetylenes and thiophenes from asteraceae on herbivorous insects

Polyacetylenes and their thiophene derivatives, characteristic secondary metabolites of the Asteraceae, were examined for their effects on herbivorous insects. Three thiophenes (a monothiophene, a bithiophene, and -terthienyl) and four polyacetylenes (phenylheptatriyne, phenylheptadiynene, phenylheptadiyene acetate, and matricaria lactone) were studied for their phototoxicity and light-independent toxicity to (1) a polyphagous lepidopteran,Ostrinia nubilalis, whose host range includes a number of phototoxic Asteraceae, (2) a polyphagous lepidoteran,Euxoa messoria, whose host range includes very few species of Asteraceae, and (3) an oligophagous lepidopteran,Manduca sexta, which is a specialist on Solanaceae. Several compounds were phototoxic toM. sexta andE. messoria even at very low irradiance levels, but behavioral adaptations, including spinning silk and boring into diet, allowedO. nubilalis to avoid photosensitization. Light-independent activity of the compounds to all three species involved feeding deterrence increasing in the orderO. nubilalis, E. messoria, andM. sexta, and longterm metabolic toxicity in the form of impaired nutrient utilization. The biosynthetically derived thiophenes were more toxic than their acetylenic precursors, and toxicity increased with increasing number of thiophene rings. The results are discussed in terms of plant-insect coevolution.     

Phytotoxic substances in root exudates of cucumber (Cucumis sativus L.)

The addition of activated charcoal to a nutrient solution for the hydroponic culture of cucumber resulted in significant increases in the dry weight of the plant and fruit yield. Hydrophobic root exudates were collected at different growth stages with Amberlite XAD-4 resin and bioassayed with lettuce seedlings. The exudates at the reproductive stage were more phytotoxic than those at the vegetative stage. The exudates contained organic acids such as benzoic,p-hydroxybenzoic, 2,5-dihydroxybenzoic, 3-phenylpropionic, cinnamic,p-hydroxycinnamic, myristic, palmitic, and stearic acids, as well asp-thiocyanatophenol and 2-hydroxybenzothiazole, all of which, except 2-hydroxybenzothiazole, were toxic to the growth of lettuce.     

Valorisation of hardwood hemicelluloses in the kraft pulping process by using an integrated biorefinery concept

A primary hydrolysis treatment (auto or acid-catalysed) of Eucalyptus globulus wood was performed before the cooking stage to extract part of the hemicelluloses that otherwise would be dissolved in the kraft liquor and burned. As xylose was the main monosaccharide Pichia stipitis was selected to produce bioethanol. Two methods were tested, with different alkalis, to reduce hydrolysates toxicity and adjust pH. A two-step method using Ca(OH)₂ leads to better fermentation results. Acid hydrolysates promoted higher ethanol concentrations (12 g L⁻¹) with high productivity and yield values (0.22 g_eth L⁻¹h⁻¹ and 0.48 g_eth/g_{xyl eqs}), whilst auto-hydrolysates, even after a secondary hydrolysis, gave low ethanol concentrations (2–4 g L⁻¹). The impacts on kraft cooking and pulp quality were also studied in order to fully understand the feasibility of this biorefinery concept (combining ethanol production and hardwood pulping). A decrease of the overall pulp yield (10% for auto- and 15% for acid-hydrolysis) was observed. However, a decrease on bleaching requirements (up to 15%) and on brightness reversion was registered. Moreover, auto-hydrolysis improves pulp viscosity, whilst acid-hydrolysis is more efficient in metals leaching. Overall, from the pulp production point of view, auto-hydrolysis conditions are more attractive than the acid-catalysed ones.     

Evidence for allelochemical attraction of the coffee berry borer,Hypothenemus hampei,by coffee berries

Petri dish choice tests conducted on the coffee berry borer (CBB),Hypothenemus hampei, showed that females were able to discriminate between coffee berries at different ripening stages. A Y-shaped glass olfactometer was used to demonstrate that coffee berries emitted volatile chemicals that elicited upwind movement by female CBB. Olfactometer tests with three different solvent extracts of berries showed that at least some of the attractive chemical(s) released by the coffee berries could be extracted with acetone.     

Inheritance Of Resistance to Mammalian Herbivores and of Plant Defensive Chemistry in an <Emphasis Type="Italic">Eucalyptus</Emphasis> Species

Hybridization in plants provides an opportunity to investigate the patterns of inheritance of hybrid resistance to herbivores, and of the plant mechanisms conferring this resistance such as plant secondary metabolites. We investigated how inter-race differences in resistance of Eucalyptus globulus to a generalist mammalian herbivore, Trichosurus vulpecula, are inherited in their F₁ hybrids. We assessed browsing damage of 3-year-old trees in a common environment field trial on four hybrid types of known progeny. The progeny were artificial intra-race crosses and reciprocal inter-race F₁ hybrids of two geographically distinct populations (races) of E. globulus north-eastern Tasmania and south-eastern Tasmania. Populations of trees from north-eastern Tasmania are relatively susceptible to browsing by T. vulpecula, while populations from south-eastern Tasmania are more resistant. We assessed the preferences of these trees in a series of paired feeding trials with captive animals to test the field trial results and also investigated the patterns of inheritance of plant secondary metabolites. Our results demonstrated that the phenotypic expression of resistance of the inter-race F₁ hybrids supported the additive pattern of inheritance, as these hybrids were intermediate in resistance compared to the pure parental hybrids. The expression of plant secondary metabolites in the F₁ hybrids varied among major groups of individual compounds. The most common pattern supported was dominance towards one of the parental types. Together, condensed tannins and essential oils appeared to explain the observed patterns of resistance among the four hybrid types. While both chemical groups were inherited in a dominant manner in the inter-race F₁ hybrids, the direction of dominance was opposite. Their combined concentration, however, was inherited in an additive manner, consistent with the phenotypic differences in browsing.     

Bioactive Steroids and Triterpenes from Melilotus messanensis and Their Allelopathic Potential

The aerial parts of Melilotus messanensis (sweet clover) afforded, from moderately and polar bioactive fractions, 11 triterpenes and five steroids. A series of aqueous solutions at 10^–4–10^–9 M of five steroids and nine triterpenes was monitored to test their effects on germination and growth of the monocots Hordeum vulgare and Allium cepa, and the dicots Lactuca sativa (var. nigra and var. romana) and Lycopersicon esculentum. An important stimulatory effect on H. vulgare germination (between 40% and 80% for almost all tested compounds) was observed. Some considerations about the ecological role of triterpenes on M. messanensis are made. In addition to known lupane triterpenes (platanic acid and 3,28,30-lup-20(29)-enetriol), oleanane triterpenes (soyasapogenol B, soyasapogenol G, and messagenolide), a gammacerane triterpene (gammacer-16-en-3-one), five new lupane triterpenes (messagenic acids A–E: (27-cis-4-hydroxycinnamoyloxy)-betulinic acid; 27-(trans-4-hydroxycinnamoyloxy)betulinic acid; 20(S)-3-hydroxy-29-oxolupan-28-oic acid; 3,30-dihydroxylup-20(29)-en-28-oic acid; and 3,20-dihydroxylup-18(19)-en-28-oic acid, respectively), and sterols (-sitosterol, ergosterol peroxide, 7-hydroxysitosterol, 7-hydroxysitosterol, and 7-oxositosterol) were isolated and chemically characterized. Their structures and stereochemistry were elucidated by spectroscopic methods (one- and two-dimensional ¹H and ¹³C NMR, IR, MS).     

Development of a bioassay to test phloem sap samples from lettuce for resistance toNasonovia ribisnigri (homoptera,aphididae)

TheNr-gene-based resistance of lettuce to the aphidNasonovia ribisnigri (Mosley) has previously been shown to be located in the phloem. Since chemical analyses of the phloem sap had shown no differences between resistant and susceptible lines, a bioassay was developed in order to test samples from resistant and susceptible plants on aphid feeding. For this, whole-plant extracts, honeydew, and EDTA-collected phloem extracts were obtained, and a sensitive bioassay was developed using EDTA samples. The EDTA was removed, and samples were added to a simple sucrose solution or to a complex artificial diet and presented in a choice situation comparing extracts from resistant and susceptible plants. EDTA-collected phloem sap samples from susceptible plants were preferred to those from resistant plants. The resistance is probably based on a feeding deterrent activity of the phloem sap in the resistant plant.     

Mimosine-inhibited seed germination,seedling growth,and enzymes ofOryza sativa L

The effect of mimosine (50 ppm and 100 ppm concentrations) onOryza sativa (rice) seed germination; root and shoot growth, i.e., length and fresh weight of seedlings; activities of nitrate reductase, peroxidase, catalase, and IAA oxidase were investigated. Significant inhibition in seed germination and shoot length was noted. Root length was inhibited by 100 ppm mimosine; however, the 50 ppm was not significant. Root and shoot fresh weight was not significantly inhibited by the tested concentrations of mimosine. Significant inhibition in activities of nitrate reductase, peroxidase and its isoenzymes, catalase, and IAA oxidase was observed. Ecophysiological implications of mimosine phytotoxicity are discussed.     

Chemoattraction ofBiomphalaria glabrata (Gastropoda: Planorbidae) to lipid standards and lipophilic factors in leaf lettuce and Tetramin

Chemoattraction of individualBiomphalaria glabrata snails for lipid standards and lipophilic fractions of leaf lettuce and Tetramin were studied in a Petri dish bioassay. Snails were more significantly attracted to a whole Tetramin lipophilic fraction than that of leaf lettuce. Thin-layer chromatography showed that major neutral lipid fractions in Tetramin were triacylglycerols, free fatty acids, and free sterols, and in leaf lettuce were free fatty acids and a mixed free sterol-chlorophyll fraction. Snails were significantly attracted to both the free fatty acid and free sterol fractions from Tetramin, but only to the free fatty acid fraction from leaf lettuce. Snails were significantly attracted to a mixed lipid standard containing equal amounts of phosphatidylcholine, cholesterol, oleic acid, triolein, and cholesteryl oleate. Of four individual neutral lipid standards tested, i.e., cholesterol, oleic acid, triolein, and cholesteryl oleate, snails were only attracted to cholesteryl oleate.     

Chemical Stimulants of Leaf-Trenching by Cabbage Loopers: Natural Products, Neurotransmitters, Insecticides, and Drugs

Larvae of the cabbage looper, Trichoplusia ni (Lepidoptera: Noctuidae), often transect leaves with a narrow trench before eating the distal section. The trench reduces larval exposure to exudates, such as latex, during feeding. Plant species that do not emit exudate, such as Plantago lanceolata, are not trenched. However, if exudate is applied to a looper's mouth during feeding on P. lanceolata, the larva will often stop and cut a trench. Dissolved chemicals can be similarly applied and tested for effectiveness at triggering trenching. With this assay, I have documented that lactucin from lettuce latex (Lactuca sativa), myristicin from parsley oil (Petroselinum crispum), and lobeline from cardinal flower (Lobelia cardinalis) elicit trenching. These compounds are the first trenching stimulants reported. Several other constituents of lettuce and parsley, including some phenylpropanoids, monoterpenes, and furanocoumarins had little or no activity. Cucurbitacin E glycoside found in cucurbits, another plant family trenched by cabbage loopers, also was inactive. Lactucin, myristicin, and lobeline all affect the nervous system of mammals, with lobeline acting specifically as an antagonist of nicotinic acetylcholine receptors. To determine if cabbage loopers respond selectively to compounds active at acetylcholine synapses, I tested several neurotransmitters, insecticides, and drugs with known neurological activity, many of which triggered trenching. Active compounds included dopamine, serotonin, the insecticide imidacloprid, and various drugs such as ipratropium, apomorphine, buspirone, and metoclopramide. These results document that noxious plant chemicals trigger trenching, that loopers respond to different trenching stimulants in different plants, that diverse neuroactive chemicals elicit the behavior, and that feeding deterrents are not all trenching stimulants. The trenching assay offers a novel approach for identifying defensive plant compounds with potential uses in agriculture or medicine. Cabbage loopers in the lab and field routinely trench and feed on plants in the Asteraceae and Apiaceae. However, first and third instar larvae enclosed on Lobelia cardinalis (Campanulaceae) failed to develop, even though the third instar larvae attempted to trench. Trenching ability does not guarantee effective feeding on plants with canal-borne exudates. Cabbage loopers must not only recognize and respond to trenching stimulants, they must also tolerate exudates during the trenching procedure to disable canalicular defenses.     

Phloem sap collection from lettuce (Lactuca sativa L.): Methodology and yield

Three methods to collect phloem sap on different lettuce lines were optimized and are described in detail. The success ratio for stylectomy of aphids was over 80% through the combination of a specially designed setup and electrical penetration graphs to monitor phloem sap ingestion. For unknown reasons on some lettuce lines stylets never showed sustained exudation. There were clear differences in stylet exudation between two aphid species on the same lettuce line. Honeydew collection in hexadecane made accurate quantitative analysis possible; samples were large and clean, but biotransformed. The EDTA chelation method produced large samples, but dilution, oxidation, and impurities from the wound surface reduced the reliability.     

Isolation and Structural Characterization of a Water-Soluble Germination Inhibitor from Scotch Thistle (Onopordum acanthium) Cypselas

Cypsela dormancy in Scotch thistle (Onopordum acanthium) may be affected by the presence of chemical inhibitors. To investigate this phenomenon, a leachate from O. acanthium cypselas was tested for its ability to inhibit germination of the cypselas from which it was derived (i.e., autoinhibition). Leachates varied in their degree of autoinhibition, depending on the cypsela population from which they were prepared. Overall, removal of leachate from a group of O. acanthium cypselas increased their germinability. Using lettuce (Lactuca sativa) cypselas as an indicator species, bioactivity-guided fractionation was used to isolate a water-soluble, para-substituted benzamide from O. acanthium cypselas, which caused germination inhibition. Various chromatographic, spectroscopic, and spectrometric techniques were applied to the characterization of the bioactive compound.