Chemistry Of Allelopathy

As we know, plant and microbial compounds are continuously analyzed as potential sources of herbicides, pesticides, and pharmaceuticals because they provide a diversity of carbon skeletons and there has been success in that a number of compounds have shown biological activity. The same bioassays and techniques to reveal mechanisms of action apply to the search for herbicides as in the study of allelopathy. Certainly there is overlap in goals and compounds studied, but there also is a difference in that the starting point in the 'herbicide search' might be any natural product, as opposed to one identified with allelopathy. Most inhibitors of plants are secondary compounds that have their origin in either the shikimate or acetate pathways, or they are compounds having skeletal components from both of these origins (Einhellig, 2002). Waller et al. (1999) listed over twenty classes of secondary metabolites that are produced, stored, and released into the rhizosphere where they have biological activity as well as undergo microbial transformation and degradation. Einhellig (2002) concluded that the 14 categories suggested by Rice (1984) are sufficiently broad to still retain validity: water-soluble organic acids, straight-chain alcohols, aliphatic aldehydes and ketones, unsatured lactones, long-chain fatty acids and polyacetylenes, naphthoquinones, anthraquinones and complex quinones, gallic acids and polyphloroglucinols, cinnamic acids, coumarins, flavonoids, tannins, terpenoids, and steroids, amino acids, and purines and nucleosides.

in this chapter some of the main compounds and studies associated with allelopathy will be mentioned.

Terpenoids and phenolics are the most common compounds involved in allelo-pathic interactions. Terpenoids are the largest group of plant chemicals (15,000-20,000) with a common biosynthethic origin. The terpenoid pathway generates great structural diversity and complexity of compounds, thus generating enormous potential for mediating ecological interactions (Duke, 1991; Langenheim, 1994). Terpenoids may produce effects on seeds and soil microbiota through volatilization, leaching from plants, or decomposition of plant debris. These interactions can significantly affect community and ecosystem properties, although studies of plant-plant chemical interactions have often been controversial because of difficulty in unambiguously demonstrating interference by chemical inhibition rather than through resource competition or other mechanisms (Harper, 1977).

3.1. Terpenoids and sesquiterpene lactones

Vokou et al. (2003) compared the potential allelopathic activity of 47 monoterpenoids 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. These authors 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 effect of the sesquiterpene cacalol and extracts (water and petroleum ether) derived from the roots of Psacalium decompositum (Asteraceae) on the germination and radicle growth of two plants, Amaranthus hypochondriacus (Amaranthaceae) and Echinochloa crus-galli (Poaceae), and the radial growth of four phytopathogenic fungi was described (Anaya et al., 1996). The activity of two cacalol derivatives (methyl cacalol and cacalol acetate) was also investigated. Germination of A. hypochondriacus was inhibited by almost all the treatments. The extracts and cacalol produced a significant inhibition of radicle growth of A. hypochondriacus and E. crus-galli. cacalol acetate showed a specific inhibition on E. crus-galli, and methyl cacalol inhibited significantly the growth of A. hypochondriacus. In general, antifungal activity depended upon the target fungi and the concentration of each treatment. cacalol had also effects on the morphology and coloration of the fungal mycelium. The bioactivity shown by the extracts of Psacalium decompositum on the tested seeds and fungi is mainly due to their content in cacalol.

The allelochemical potential of Callicarpa acuminata (Verbenaceae) was investigated using a biodirected fractionation study as part of a long-term project to search for bioactive compounds among the rich biodiversity of plant communities in the Ecological Reserve El Eden, Quintana Roo, Mexico. Aqueous leachate, chloroformmethanol extract, and chromatographic fractions of the leaves of the plant species inhibited the root growth of Amaranthus hypochondriacus, Echinochloa crus-galli, and tomato (23% , 59%, and 70% respectively). some of these treatments caused a moderate inhibition of the radial growth of two phytopathogenic fungi, Helminthosporium longirostratum and Alternaria solani (18% to 31%). The chloroform-methanol (1:1) extract prepared from the leaves rendered five compounds:

isopimaric acid, a mixture of two diterpenols: sandaracopimaradien-19-ol and akhdarenol, a-amyrin, and the flavone salvigenin. The phytotoxicity exhibited by several fractions and the full extract almost disappeared when pure compounds were evaluated on the test plants, suggesting a synergistic or additive effect. Akhdarenol, a-amyrin and isopimaric acid methyl ether had antifeedant effects on Leptinotarsa decemlineata. Alpha-amyrin was most toxic to this insect. No correlation was found between antifeedant and toxic effects on this insect, suggesting that different modes of action were involved. All the test compounds were cytotoxic to insect Sf9 cells while salvigenin, akhdarenol, and isopimaric acid also affected mammalian Chinese Hamster ovary (CHo) cells. Alpha-amyrin showed the strongest selectivity against insect cells (Anaya et al., 2003). In this study the authors emphasized that allelochemicals involved in allelopathic interactions often have multiple functionality.

Sesquiterpene lactones (SL) occur in over 15 plant families, predominantly in the Asteraceae, and represent with about 3,500 naturally occurring compounds, one of the largest groups of natural products. It has been demonstrated that some sesquiterpene lactones exhibit a broad spectrum of biological activities including phytotoxic and plant growth regulatory properties, cytotoxicity and antitumor properties, antimicrobial, insecticidal, molluscicidal and antimalarial activity (Fischer, 1986). Phytotoxic terpenoids and their possible involvement in allelopathy were covered in reviews on mono- and sesquiterpenes (Evenari, 1949; Fischer, 1986, 1991, 1994) and biological activities of SL were reviewed by Stevens and Merrill (1985), Picman (1986) and Elakovich (1988). Seedlings of Ambrosia cumanensis are inhibited by leachates of the adult plants and residues in soils. Some SL of this species have been implicated in this autototoxic mechanism (Anaya and del Amo, 1978). In the same way, parthenin and coronopilin of Parthenium hysterophorus also exhibited autotoxicity toward seedlings and older plants, this fact possibly reveal a mechanism of intraspecific population regulation (Picman and Picman, 1984). Axivalin and tomentosin from the seeds of Iva axillaris were inhibitory toward the germination and growth of Abutilon theophrasti (velvetleaf) (Spencer et al., 1984). The germacranolide-type SL represented by dihydrotartridin B significantly inhibited the root growth of Brassica rapa var. pervidis (Sashida et al., 1983). The a-methylene-y-lactone group is present in many of the isolated natural sesquiterpene lactones, and has been proposed as one of the factors which can determine their allelopathic activity, in particular, as well as their biological activity in general. The different spatial arrangements that a molecule of SL can adopt is the other factor that has been related with the potential allelopathic activity of this type of secondary compounds (Macias et al., 1992). Data of several studies on the allelopathic potential of SL clearly demonstrated that they can selectively promote or inhibit germination or growth at concentrations as low as 1 |M. It is reasonable to assume that rain washes transport SL from the source plant or decomposing litter into the soil where they can reach significant concentration levels. In the case of isoalantolactone, it has been demonstrated that it can persist in mineral and organic soil for 3 months, supporting the assumption that SL play a significant role in allelopathic interactions in the environment (del Amo and Anaya, 1976; Stevens and Merril, 1985; Picman, 1986; Fischer, 1991).

Dehydrozaluzanin C, a natural SL, is a weak plant growth inhibitor with an I50 (or IC50, the concentration required to inhibit plant growth 50 %) of about 0.5 mM for lettuce root growth. It also causes rapid plasma membrane leakage in cucumber cotyledon discs. Dehydrozaluzanin C is more active at 50 |M than the same concentration of the herbicide acifluorfen. Symptoms include plasmolysis and the disruption of membrane integrity is not light dependent. Reversal of its effects on root growth was obtained with treatment by various amino acids, with histidine and glycine providing ca. 40% reversion. The strong reversal effect obtained with reduced glutathione is due to cross-reactivity with DHZ and the formation of mono- and di-adducts. Photosynthetic, respiratory and mitotic processes, as well as NADH oxidase activity appear to be unaffected by this compound. Dehydrozaluzanin C exerts its effects on plants through two different mechanisms, only one of which is related to the disruption of plasma membrane function (Galindo et al., 1999).

A structure-activity study to evaluate the effect of the trans,trans-germacranolide SL lactones costunolide, parthenolide, and their 1,10-epoxy and 11,13-dihydro derivatives (in a range of 100-0.001 |M) on the growth and germination of several mono and dicotyledon target species was carried out by Macias et al. (1999). These compounds appear to have more selective effects on the radicle growth of monocotyledons. Certain factors such as the presence of nucleophile-acceptor groups and their accessibility enhance the inhibitory activity. The levels of radicle inhibition obtained with some compounds on wheat are totally comparable to those of commercial herbicide Logran and allow proposing them as lead compounds. In addition, a structure-activity study to evaluate the effect of 17 guaianolide SL (in a range of 100-0.001 |M) on the growth and germination of several mono- and dicotyledon target species was also performed by Macias et al. (2002a). These compounds appear to have deeper effects on the growth of either monocots or dicots than the previously tested germacranolides. Otherwise, the lactone group seems to be necessary for the activity, though it does not necessarily need to be unsaturated. However, the presence of a second and easily accessible unsaturated carbonyl system greatly enhances the inhibitory activity. Lipophilicity and the stereochemistry of the possible anchoring sites are also crucial factors for the activity.

The dichloromethane extract of dried leaves of Helianthus annuus has yielded, in addition to the known SL annuolide E and leptocarpin, and the sesquiterpenes heliannuols A,C,D,F,G,H,1, the new bisnorsesquiterpene, annuionone E, and the new sesquiterpenes heliannuol L, helibisabonol A and helibisabonol B. Structural elucidation was based on extensive spectral (one and two-dimensional NMR experiments) and theoretical studies. The sesquiterpenes heliannuol A and helibisabonol A and the SL leptocarpin inhibited the growth of etiolated wheat coleoptiles (Macias et al., 2002b). In addition to (+)-, (-)- and (±)-heliannuol E, growth-inhibitory activities of five synthetic chromanes and four tetrahydrobenzo[b]oxepins were examined against oat and cress. All heliannuol E isomers exhibited similar biological activities against cress, whereas when tested against oat roots, the unnatural optical isomer (+) showed no inhibitory activity. Four brominated chromans and two tetrahydrobenzo[b]oxepins derivatives also showed apparent inhibition against both cress and oat (Doi et al., 2004).

The tremendous impact of parasitic plants on world agriculture has prompted much research aimed at preventing infestation. Orobanche and Striga spp. are two examples of parasitic weeds that represent a serious threat to agriculture in large parts of the world. The life cycle of these parasitic weeds is closely regulated by the presence of their hosts, and secondary metabolites that are produced by host plants play an important role in this interaction. A special interest has been arising on those host-produced stimulants that induce the germination of parasite seeds. Three classes of compounds have been described that have germination-stimulating activity: dihydrosorgoleone, the strigolactones and SL. Keyes et al. (2001) suggest that dihydrosorgoleone is the active stimulant in the root exudates of sorghum and other monocotyledonous hosts. However, Butler et al. (1995) and Wigchert et al. (1999) suggest that dihydrosorgoleone is less likely to be the germination stimulant in vivo because of its low water solubility, and because no correlation between its production and the germination of Striga has been found. To date, there is no definite proof that the germination of parasitic weed seeds in the field is induced by one single signal compound or class of compounds (and indeed such proof will be hard to obtain) (Bouwmeester et al., 2003). The capacity of SL, which share some structural features with the strigolactones, to induce the germination of S. asiatica has been reported (Fischer et al., 1989, 1990). In addition, a decade after the results of Fischer studies, Francisco Macías and his group (Pérez de Luque et al., 2000; Galindo et al., 2002) performed some studies of the structure-activity relationship (SAR) directed to evaluate the effect of several SL as germination stimulants of three Orobanche spp. (O. cumana, O. crenata, and O. ramosa). Results are compared with those obtained in the same bioassay with an internal standard, the synthetic analogue of strigol GR-24. A high specificity in the germination activity of SL on the sunflower parasite O. cumana has been observed, and a relationship between such activity and the high sunflower SL content is postulated. Molecular properties of the natural and synthetic germination stimulants (GR-24, GR-7, and Nijmegen-1) and SL have been studied using MMX and PM3 calculations. Consequently, comparative studies among all of them and their activities have been made. SL tested present similarities in molecular properties such as the volume of the molecule and the spatial disposition of the carbon backbone to the natural germination stimulant orobanchol. These properties could be related to their biological activity. Considering that the sun-flower-O. cumana interaction is highly specific and that sunflower contains many SL, it is tempting to speculate that O. cumana has evolved to respond to sesquiterpene lactones (and not or less to strigolactones) (Bouwmeester et al., 2003).

3.2. Phenolics

In relation with phenolics, Inderjit et al. (1997) conducted a study to understand the effects of certain phenolics, terpenoides, and their equimolar mixture through agar gel and soil growth bioassays and their recovery from soils. The eight compounds selected for this study were p-hydroxybenzoic acid, ferulic acid, umbelliferone, catechin, emodin, 1,8-cineole, carvone, and betulin. Lettuce (Lactuca sativa L.) was used as test species for agar gel and soil growth bioassays. Root and shoot growth of lettuce was inhibited for all the above except emodin and catechin. However, in soils treated with different phenolics and terpenoids, only root growth of lettuce was inhibited, whereas shoot growth was promoted. Recovery of p-hydroxybenzoic acid and umbelliferone was higher in unautoclaved soils, while that of catechin was lower.

Nava-Rodriguez et al. (in press) observed the in vitro effects of aqueous leachates from fresh and dry, flowering and vegetative stage of Phaseolus species, faba bean, alfalfa, vetch, maize, and squash, and weed species on the root growth of selected crop and weeds, as well as on two strains of Rhizobium leguminosarum biovar phaseoli (CPMex1 and Tlaxcala). Most of the specimens were collected in a traditional agricultural drained field ("Camellon") in Tlaxcala, Mexico where maize, beans, squash, alfalfa, faba-beans, and vetch are cultivated in mixed or rotation crops. significant effects of leachates from fresh vegetative and flowering cultivated plants and weeds were predominantly stimulatory on the growth of tested crops, being the leachates from fresh aerial parts of alfalfa and pinto bean the most stimulatory. Nevertheless, aqueous leachates from fresh and dry cultivated legumes (vegetative and flowering) inhibited the growth of weeds. in contrast, the aqueous leachates from the dry aerial part of almost all plants resulted inhibitory on the root growth of the test crops, except maize. Aqueous leachates were also evaluated on the growth of two strains of Rhizobium leguminosarum biovar phaseoli. Leachates from some of the tested crops significantly stimulated the growth of both Rhizobium strains. The aqueous leachates from fresh aerial parts of the weeds Simsia amplexicaulis and Tradescantia crassifolia significantly inhibited the growth of CPMex1 Rhizobium strain. On the other hand, the aqueous leachates from fresh roots of these same weeds inhibited the growth of the Tlaxcala strain. in preliminary chemical tests using thin layer chromatography (TLC), phenolics were detected in dry aerial parts of vegetative alfalfa, pinto bean, and vetch, and dry aerial part of flowering faba bean suggesting the role of these compounds in the allelopathic effects of these legumes.

Nilsson et al. (1998) reported on the temporal variation of phenolics and a dihydrostilbene, batatasin III, in Empetrum hermaphroditum leaves. These authors reported that first year shoots produced higher levels of phenolics than older tissues. High phenolic concentration was maintained through the second year, but it declined afterwards. However, the phytotoxicity of E. hermaphroditum extracts was related more to batatasin III than phenolics.

Hyder et al. (2002) performed a study focused on the presence and distribution of secondary phenolic compounds found within creosotebush (Larrea tridentata). Total phenolics, condensed tannins and nordihydroguaiaretic acid (NDGA) were measured in nine categories of tissue within creosotebush. Total phenolic and condensed tannin concentrations were determined using colorimetric methods while NDGA content was determined with high performance liquid chromatography (HPLC). Phenolics were present throughout the plant with the highest concentrations in green stems (40.8 mg/g), leaves (36.2 mg/g), and roots (mean for all root categories=28.6 mg/g).

Condensed tannins were found in all tissues with highest concentrations in flowers (1.7 mg/g), seeds (1.1 mg/g), and roots less than 5 mm in diameter (1.1 mg/g). Flowers, leaves, green stems and small woody stems (<5 mm in diameter) all contained NDGA with highest concentrations in leaves (38.3 mg/g) and green stems (32.5 mg/ g).

Another study conducted by Singh et al. (2003a) assessed the phytotoxicity of Ageratum conyzoides, a weed of cultivated areas, to the growth and establishment of wheat (Triticum aestivum). The lengths of the radicle and coleoptile and the seedling dry weight of wheat were significantly reduced when wheat was grown in field soil previously infested with A. conyzoides, compared to control soil collected from an area devoid of this weed. Even extracts prepared from A. conyzoides soil were inhibitory, indicating the presence of some water-soluble phytotoxins in the soil. To determine the possible contribution of the weed in releasing these phytotoxins, growth studies involving leaf residues and their extracts and amended soils (prepared by incorporating leaf residues and residue extracts) were also performed on wheat. With all treatments, an inhibitory effect of A. conyzoides was found, compared to respective controls. A significant amount of water-soluble toxic phenolics was found to be present in the soil infested with A. conyzoides, leaf residues and the amended soils. The amount of phenolics correlated well with growth performance in the respective treatments (see chapter 11).

Aqueous leachates of roots of the perennial weed Pluchea lanceolata, its root-incorporated soil and rhizosphere soil, interfered with the seedling growth of certain plant species. The soils from the rhizosphere zone of this plant had significantly higher total phenolics and HPLC analysis revealed that phenolic fractions represented by retention times of 1.6, 1.9, 2.5 (simple phenol, chlorogenic acid and phloroglucinol respectively), 3.7 and 4.3 min were contributed by roots of the weed to the soil. The phenolic fraction represented by the retention time 3.3 (formononetin 7-O-glucoside) was detected in the weed's rhizosphere soils and not in the root-incorporated soils. UV spectral studies established the presence of phloroglucinol, simple phenol, chlorogenic acid, formononetin 7-O-glucoside, and methylated coumarins in the root leachate, which affect the seedling growth of mustard (Brassica juncea) (Inderjit and Dakshini, 1994).

The effects of five phenolic compounds, catechol, protocatechuic, p-coumaric, p-hydroxybenzoic, ferulic acids and their mixture were studied on pH, organic matter, organic-nitrogen, total phenolic content and certain inorganic ions of forest mineral soils (Ae and B horizons). The A- and B-horizon soils, were amended with 10-4 M concentration of each phenolic compound and their mixture. In general, soil properties were affected by phenolics amendement. However, soils amended with catechol did not influence any of the soil characteristics. Contents of organic matter, nitrogen and phosphate were lower in soils amended with different phenolic compounds compared to the unamended control soil (Inderjit and Mallik, 1997).

Low molecular weight phenolic compounds have been identified in fresh leaves and in soils in which leaves of five varieties of Capsicum annuum were decomposing. Six phenolic compounds were tested in laboratory bioassays for their allelopathic effects on germination and seedling growth of six weeds. Ferulic acid, gallic acid, p-coumaric acid, p-hydroxybenzoic acid, vanillic acid, and p-vanillin were bioassayed in concentrations of 10, 1, 0.1, and 0.01 mM. Equimolar mixtures containing all these phenolics were prepared at the final total concentration of 10, 1, 0.1, and 0.01 mM to test for possible interactive effects. Chenopodium album, Plantago lanceolata, Amaranthus retroflexus, Solanum nigrum, Cirsium sp. and Rumex crispus were the selected target weeds. The highest concentration of the compounds inhibited the germination of all these weeds, but lower concentrations had no effect or were stimulatory. However, effects varied with the weed species, the concentration of the compound tested and the compound itself. In assays with the mixture of phenolics some additive effects were found (Reigosa et al., 1999).

Reversible sorption of phenolic acids by soils may provide some protection to phenolic acids from microbial degradation. In the absence of microbes, reversible sorption 35 days after addition of 0.5-3 mu mol/g of ferulic acid or p-coumaric acid was 8-14% in Cecil A(p) horizon and 31-38% in Cecil B-t horizon soil materials. The reversibly sorbed/solution ratios (r/s) for ferulic acid or p-coumaric acid ranged from 0.12 to 0.25 in A(p) and 0.65 to 0.85 in B-t horizon soil materials. When microbes were introduced, the r/s ratio for both the A(p) and B-t horizon soil materials increased over time up to 5 and 2, respectively, thereby indicating a more rapid utilization of solution phenolic acids over reversibly sorbed phenolic acids. The increase in r/s ratio and the overall microbial utilization of ferulic acid and/or p-coumaric acid were much more rapid in A(p) than in B-t horizon soil materials. Reversible sorption, however, provided protection of phenolic acids from microbial utilization for only very short periods of time. Differential soil fixation, microbial production of benzoic acids (e.g., vanillic acid and p-hydroxybenzoic acid) from cinnamic acids (e.g., ferulic acid and p-coumaric acid, respectively), and the subsequent differential utilization of cinnamic and benzoic acids by soil microbes indicated that these processes can substantially influence the magnitude and duration of the phytoxicity of individual phenolic acids (Blum, 1998).

Soil solution concentrations of allelopathic agents (e.g., phenolic acids) estimated by soil extractions differ with extraction procedure and the activities of the various soil sinks (e.g., microbes, clays, organic matter). This led to the hypothesis that root uptake of phenolic acids is a better estimator of dose than soil solution concentrations based on soil extracts. This hypothesis was tested by determining the inhibition of net phosphorus uptake of cucumber seedlings treated for 5 hr with ferulic acid in whole-root and split-root nutrient culture systems. Experiments were conducted with II ferulic acid concentrations ranging from 0 to 1 mM, phosphorus concentrations of 0.25, 0.5, or 1 mM, and solution pH values of 4.5, 5.5, or 6.5 applied when cucumber seedlings were 9, 12, or 15 days old. The uptake or initial solution concentration of ferulic acid was regressed on ferulic acid inhibition of net phosphorus uptake. Attempts were made to design experiments that would break the collinearity between ferulic acid uptake and phosphorus uptake. The original hypothesis was rejected because the initial ferulic acid solution concentrations surrounding seedling roots were more frequently and consistently related to the inhibition of net phosphorus uptake than to ferulic acid uptake by these roots. The data suggest that root contact, not uptake, is responsible for the inhibitory activity of phenolic acids (Lehman and Blum, 1999).

Bulk-soil and rhizosphere bacteria are thought to exert considerable influence over the types and concentrations of phytotoxins, including phenolic acids that reach a root surface. Induction and/or selection of phenolic acid-utilizing (PAU) bacteria within the bulk-soil and rhizosphere have been observed when soils are enriched with individual phenolic acids at concentrations greater than or equal to 0.25 |imol/g soil. However, since field soils frequently contain individual phenolic acids at concentrations well below 0.1 | mol/g soil, the actual importance of such induction and/or selection remains uncertain. Common bacteriological techniques (e.g., isolation on selective media, and plate dilution frequency technique) were used to demonstrate in Cecil Ap soil systems: (i) that PAU bacterial communities in the bulk soil and the rhizosphere of cucumber seedlings were induced and/or selected by mixtures composed of individual phenolic acids at concentrations well below 0.25 |imol/g soil; (ii) that readily available carbon sources other than phenolic acids, such as glucose, did not modify induction and/or selection of PAU bacteria; (iii) that the resulting bacterial communities readily utilize mixtures of phenolic acids as a carbon source; and (iv) that depending on conditions (e.g., initial PAU bacterial populations, and phenolic acid concentration) there were significant inverse relationships between PAU bacteria in the rhizosphere of cucumber seedlings and absolute rates of leaf expansion and/or shoot biomass. The decline in seedling growth could not be attributed to resource competition (e.g., nitrogen) between the seedlings and the PAU bacteria in these studies. The induced and/or selected rhizosphere PAU bacteria, however, reduced the magnitude of growth inhibition by phenolic acid mixtures. For a 0.6 |imol/g soil equimolar phenolic acid mixture composed of p-coumaric acid, ferulic acid, p-hydroxybenzoic acid, and vanillic acid, modeling indicated that an increase of 500% in rhizosphere PAU bacteria would lead to an approximate 5% decrease (e.g., 20-25%) in inhibition of absolute rates of leaf expansion (Blum et al., 2000).

Allelopathy due to humus phenolics is a cause of natural regeneration deficiency in subalpine Norway spruce (Picea abies) forests. If inhibition of spruce germination and seedling growth due to allelochemicals is generally accepted, in contrast there is a lack of knowledge about phenolic effects on mycorrhizal fungi. Thus, Souto et al. (2000) tested the effects of a humic solution and its naturally occurring phenolics on the growth and respiration of two mycorrhizal fungi: Hymenoscyphus ericae (symbiont of Vaccinium myrtillus, the main allelochemical-producing plant) and Hebeloma crustuliniforme (symbiont of P. abies, the target plant). Growth and respiration of H. crustuliniforme were inhibited by growth medium with the original humic solution (6% and -30%), respectively, whereas the same humic solution did not affect growth but decreased respiration of H. ericae (-55%). When naturally occurring phenolics (same chemicals and concentrations in the original humic solution) were added to the growth medium, growth of H. crustuliniforme was not affected, whereas that of H. ericae significantly increased (+10%). These authors concluded that H. ericae is better adapted to the allelopathic constraints of this forest soil than H. crustuliniforme and that the dominance of V. myrtillus among understory species could be explained in this way.

Inderjit and Duke (2003) mentioned that the best evidence for allelopathy should include some understanding of natural concentrations and rates of allelochemicals. For example, (±)-catechin has been isolated from Centaurea maculosa (Bais et al., 2002), an invasive species in North America for which other lines of evidence suggest root allelopathy (Ridenour and Callaway, 2001). The more common enantiomer, (+)-catechin, has anti-bacterial functions, whereas (-)-catechin has strong allelopathic effects on other plants. (±)-catechin is harmless to C. maculosa, but has negative effects on other species at concentrations of ~100 mg L-1. (±)-catechin is exuded from C. maculosa roots creating concentrations from 83.2 to 185 mg L-1 in aqueous solutions. Importantly, Bais et al. (2002) found (±)-catechin in extracts from natural soils in fields containing C. maculosa in concentrations as far higher than the minimum required dose, ranging from 291.6 to 389.8 p g cm-3.

In allelopathy studies a central goal is to isolate, identify, and characterize allelochemicals from the soil. However, since it is essentially impossible to simulate exact field conditions, experiments must be designed with conditions resembling those found in natural systems. Inderjit (1996) argued that allelopathic potential of phenolics can be appreciated only when we have a good understanding of i) species responses to phenolic allelochemicals, ii) methods for extraction and isolation of active phenolic allelochemicals, and iii) how abiotic and biotic factors affect phenolic toxicity.

Duke et al. (2003) summarized the recent research of the Agricultural Research Service of United States Department of Agriculture on the use of natural products to manage pests. They discussed some studies on the use of both phytochemicals and diatomaceous earth to manage insect pests. Chemically characterized compounds, such as a saponin from pepper (Capsicum frutescens L), benzaldehyde, chitosan and 2-deixy-D-glucose are being studied as natural fungicides. Resin glycosides for pathogen resistance in sweet potato and residues of semitropical leguminous plants for nematode control are also under investigation. Bioassay-guided isolation of compounds with potential use as herbicides or herbicide leads is underway at several locations. New natural phytotoxin molecular target sites (asparagine synthetase and fructose-1,6-bisphosphate aldolase) have been discovered. Weed control in sweet potato and rice by allelopathy is under investigation. Molecular approaches to enhance allelopathy in sorghum are also being undertaken. The genes for polyketide synthases involved in production of pesticidal polyketide compounds in fungi are found to provide clues for pesticide discovery. Gene expression profiles in response to fungicides and herbicides are being generated as tools to understand more fully the mode of action and to rapidly determine the molecular target site of new, natural fungicides and herbicides.

Research on the chemical basis for allelopathy has often been hindered by the complexity of plant and soil matrices, making it difficult to track active compounds. Recent improvements in the cost and capabilities of bench-top chromatography-mass spectrometry instruments make these tools more powerful and more widely available to assist with molecular studies conducted in today's expanding field. Such instrumental techniques are herein recommended as economically efficient means of advancing the rigor of allelopathy research and assisting the development of a better understanding of the chemical basis for the allelopathy phenomenon (Haig, 2001).

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