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Asparagus (Asparagus officinalis L.) is a widely cultivated perennial veritable and can be harvested more than ten years. However, the crop quality and yield decline after a few year’s cultivation, which is called “asparagus decline”. Even though those asparagus plants were replaced with new young asparagus plants, the productivity and quality of the crop remain relatively low, which is known as a “asparagus replant problem”. One of the possible reasons for “asparagus decline” and “asparagus replant problem” is thought to be autotoxicity of asparagus. However, the compounds involved in the autotoxicity is not clear. The objective of this study was therefore to determine the potential role of autotoxicity in the “asparagus decline” and “asparagus replant problem”. An aqueous methanol extract of 10-year-asparagus-cultivated soils inhibited the growth of asparagus seedlings and other two test plants with concentration dependent manner. The result confirmed that the asparagus soils have autotoxic activity. The extract was then purified by several chromatographies with monitoring the inhibitory activity and a potent growth inhibitory substance causing the autotoxic effect was isolated. The chemical structures of the compound was determined by spectral data to be trans-cinnamic acid. trans-Cinnamic acid inhibited the growth of asparagus seedlings at concentrations greater than 10 μM. The concentrations required for 50% growth inhibition of asparagus (IC50) were 24.1–41.6 μM. trans-Cinnamic acid accumulated 174 μM in the 10-year-asparagus-cultivated soils, which may be enough levels to cause the growth inhibition on asparagus considering its IC50 value. Therefore, trans-cinnamic acid may contribute to the autotoxic effect of asparagus soils, and may be in part responsible for “asparagus decline” and “asparagus replant problem”.

Japanese red pine (Pinus densiflora Sieb. et Zucc.) forests are characterized by sparse understory vegetation although sunlight intensity on the forest floor is sufficient for undergrowth. The possible involvement of pine allelopathy in the establishment of the sparse understory vegetation was investigated. The soil of the red pine forest floor had growth inhibitory activity on six test plant species including Lolium multiflorum, which was observed at the edge of the forest but not in the forest. Two growth inhibitory substances were isolated from the soil and characterized to be 15-hydroxy-7-oxodehydroabietate and 7-oxodehydroabietic acid. Those compounds are probably formed by degradation process of resin acids. Resin acids are produced by pine and delivered into the soil under the pine trees through balsam and defoliation. Threshold concentrations of 15-hydroxy-7-oxodehydroabietate and 7-oxodehydroabietic acid for the growth inhibition of L. multiflorum were 30 and 10μM, respectively. The concentrations of 15-hydroxy-7-oxodehydroabietate and 7-oxodehydroabietic acid in the soil were 312 and 397μM, respectively, which are sufficient concentrations to cause the growth inhibition because of the threshold. These results suggest that those compounds are able to work as allelopathic agents and may prevent from the invasion of herbaceous plants into the forests by inhibiting their growth. Therefore, allelopathy of red pine may be involved in the formation of the sparse understory vegetation.

Leucas aspera (Lamiaceae), an aromatic herbaceous plant, is well known for many medicinal properties and a number of bioactive compounds against animal cells have been isolated. However, phytotoxic substances from L. aspera have not yet been documented in the literature. Therefore, current research was conducted to explore the phytotoxic properties and substances in L. aspera. Aqueous methanol extracts of L. aspera inhibited the germination and growth of garden cress (Lepidum sativum) and barnyard grass (Echinochloa crus-galli), and the inhibitory activities were concentration dependent. These results suggest that the plant may have phytotoxic substances. The extracts were then purified by several chromatographic runs. The final purification was achieved by reversed-phase HPLC to give an equilibrium (or inseparable) 3:2 mixture of two labdane type diterpenes (compounds 1 and 2). These compounds were characterized as (rel 5S,6R,8R,9R,10S,13S,15S,16R)-6-acetoxy-9,13;15,16-diepoxy-15-hydroxy-16-methoxylabdane (1) and (rel 5S,6R,8R,9R,10S,13S,15R,16R)-6-acetoxy-9,13;15,16-diepoxy-15-hydroxy-16-methoxylabdane (2) by spectroscopic analyses. A mixture of the two compounds inhibits the germination and seedling growth of garden cress and barnyard grass at concentrations greater than 30 and 3 μM, respectively. The concentration required for 50% growth inhibition (I50) of the test species ranges from 31 to 80 μM, which suggests that the mixture of these compounds, are responsible for the phytotoxic activity of L. aspera plant extract.

The grass Brachiaria brizantha, native to eastern Africa, becomes naturalized and dominant quickly in the non-native areas. It was hypothesized that phytotoxic chemical interaction between this plant and native plants may play an important role in the invasion of B. brizantha. However, no potent phytotoxic substance has been reported in this species. Therefore, we investigated possible allelopathic activity and searched for phytotoxic substances with allelopathic activity in B. brizantha. An aqueous methanol extract of B. brizantha inhibited the growth of roots and shoots of garden cress (Lepidium sativum), lettuce (Lactuca sativa), timothy (Phleum pratense) and ryegrass (Lolium multiflorum) seedlings. The extract was purified by several chromatographic runs and three allelopathically active substances were isolated and identified by spectral analysis as (6R,9R)-3-oxo-α-ionol, (6R,9S)-3-oxo-α-ionol and 4-ketopinoresinol. (6R,9R)-3-Oxo-α-ionol and (6R,9S)-3-oxo-α-ionol inhibited root and shoot growth of garden cress at concentrations greater than 30 and 10 μM, respectively. The activity of (6R,9S)-3-oxo-α-ionol was 5.3- to 6.2-fold that of (6R,9R)-3-oxo-α-ionol. The stereochemistry of the hydroxyl group at position C-9 may be important for the inhibitory activities of those compounds. 4-Ketopinoresinol inhibited root and shoot growth of garden cress at concentrations greater than 30 μM. The growth inhibitory activity of (6R,9S)-3-oxo-α-ionol was the greatest and followed by 4-ketopinoresinol and (6R,9R)-3-oxo-α-ionol. These results suggest that those phytotoxic substances may contribute to the allelopathic effect caused by B. brizantha and may be involved in the invasion of B. brizantha.

Orthosiphon stamineus (Java tea) has been widely used as traditional herb and several bioactive compounds against animal cells have been isolated. However, no bioactive compound against plants has been reported. Therefore, we investigated possible allelopathic properties and substances in O. stamineus. Aqueous methanol extracts of O. stamineus inhibited root and hypocotyl growth of cress (Lepidium sativum) and lettuce (Lactuca sativa) seedlings. Increasing the extract concentration increased the inhibition, which suggests that O. stamineus may have allelopathic properties. When the extract was divided into an ethyl acetate and an aqueous fraction, the ethyl acetate fraction showed the stronger inhibitory effect. Thus, the ethyl acetate phase was further purified, and the main allelopathic substance was isolated and identified as 13-epi-orthosiphol N, a novel compound, by spectral data. 13-epi-Orthosiphol N inhibited root and hypocotyl growth of cress and lettuce at concentrations greater than 10 μmol/L. The concentrations required for 50% inhibition ranged from 41 to 102 μmol/L. These results suggest that 13-epi-orthosiphol N may be an allelochemical and main contributor to the growth inhibitory effect of O. stamineus and may have potential as a template for the development of new plant control substances.

Asparagus ( Asparagus officinalis L.) is a perennial vegetable, but its crop productivity and quality decrease gradually. One possible reason for “asparagus decline” is thought to be the autotoxicity of asparagus. However, the autotoxic property of asparagus rhizomes remains unknown. The objective of this study was to determine the potential role of rhizomes in the autotoxicity of asparagus. An aqueous methanol extract of asparagus rhizomes inhibited the growth of asparagus seedlings and six other test plants in a concentration-dependent manners: garden cress ( Lepidum sativum L.), lettuce ( Lactuca sativa L.), alfalfa ( Medicago sativa L.), ryegrass (Lolium multiflorum Lam.), timothy ( Phleum pratense L.) and barnyardgrass ( Echinochloa crus-galli (L.) Beauv.). These results suggest that asparagus rhizomes contain autotoxic compounds. The extract was purified through several chromatographic steps with monitoring the autotoxic activity, and p -coumaric acid and iso-agatharesinol were isolated. These compounds inhibited the shoot and root growth of asparagus and two other test plants, garden cress and ryegrass, at concentrations higher than 0.1 mM. The concentrations required for 50% inhibition of the root and shoot growth of these test plants ranged from 0.36 to 0.85 mM and 0.41–1.22 mM for p -coumaric acid and iso-agatharesinol, respectively. Therefore, these compounds may contribute to the autotoxicity caused by asparagus rhizomes and may be involved in “asparagus decline”.

Aqueous methanol extracts of the traditional rice (Oryza sativa) variety Awaakamai, which is known to have the greatest allelopathic activity among Japanese traditional rice varieties, inhibited the growth of roots and shoots of cress (Lepidium sativum), lettuce (Lactuca sativa), timothy (Phleum pratense), Digitaria sanguinalis, Lolium multiflorum and Echinochloa crus-galli. Increasing the extract concentration increased the inhibition, suggesting that the extract of Awaakamai contains growth inhibitory substances. The extract of Awaakamai was purified and two main growth inhibitory substances were isolated and determined by spectral data as blumenol A and grasshopper ketone. Blumenol A and grasshopper ketone, respectively, inhibited the growth of cress shoots and roots at concentrations greater than 10 and 30 μmol/L. The concentrations required for 50% growth inhibition on cress roots and shoots were 84 and 27 μmol/L, respectively, for blumenol A, and 185 and 76 μmol/L, respectively, for grasshopper ketone. These results suggest that blumenol A and grasshopper ketone may contribute to the growth inhibitory effect of Awaakamai and may play an important role in the allelopathy of Awaakamai.

Eight cultivars of rice (Oryza sativa L.) inhibited shoot and root growth of Echinochloa crus-galli when co-cultured with rice seedlings in a bioassay medium. Momilactone A and B were found in the bioassay medium of all rice cultivars, and concentrations of momilactone A and B in the medium were 0.21-1.5 and 0.66-3.8 micromol/L, respectively, indicating that all rice cultivars may secrete momilactone A and B into the medium. Exogenously applied momilactone A and B inhibited the growth of shoots and roots of E. crus-galli at concentrations greater than 30 and 1 micromol/L, respectively. The concentrations required for 50% growth inhibition of E. crus-galli shoots and roots, respectively, were 146 and 91 micromol/L for momilactone A and 6.5 and 6.9 micromol/L for momilactone B. Considering the growth inhibitory activity and concentrations found in the bioassay medium, momilactone A may have caused only 0.8-2.2% of the observed growth inhibition of E. crus-galli roots and shoots by rice. However, momilactone B in the medium was estimated to be able to cause 59-82% of the observed growth inhibition of E. crus-galli roots and shoots by the rice seedlings. In addition, the concentrations of momilactone B in the medium reflected the observed differences in the growth inhibition of E. crus-galli by the eight rice cultivars investigated. This suggests that the allelopathic activity of rice may depend primarily on the secretion level of momilactone B. Therefore, momilactone B may play a very important role in rice allelopathy.

The secretion levels of momilactone A from rice (Oryza sativa L.) seedlings of eight cultivars into the rhizosphere were compared with the endogenous momilactone A concentrations in their shoots and roots. All rice cultivars contained momilactone A in the shoots and roots, and concentrations differed among the cultivars. Momilactone A was also found in all culture solutions in which the rice seedlings were grown, and the concentrations differed among the cultivars. The momilactone A concentrations in the culture solutions were reflected in the momilactone A concentrations in the shoots. These results suggest that all rice cultivars may produce momilactome A and secrete momilactone A into the culture solutions. The secretion levels of momilactone A may be more dependent on their capacities for momilactone A production in the shoots than on their capacities for momilactone A transportation from the shoots into the environment via the roots. As momilactone A acts as an antimicrobial and allelopathic agent, the secretion of momilactone A into the rice rhizosphere may provide a competitive advantage for root establishment through local suppression of soil microorganisms and inhibition of the growth of competing plant species.

UV-irradiation increased the concentration of momilactone B in shoots and roots of rice seedlings, and increasing the irradiation increased the concentration. The concentration in 90-min UV-irradiated shoots and roots, respectively, was 31.8- and 3.6-fold higher than that in non-irradiated shoots and roots. After UV-irradiation the concentration of momilactone B in rice shoots decreased. There was, however, an accumulation of momilactone B in the medium in which UV-irradiated seedlings were grown. Five days after UV-irradiation, momilactone B in the medium was at a level 2.5 times greater than on day 0, which was 47% of momilactone B in the seedlings, suggesting that rice may actively secrete momilactone B into medium. Therefore, UV-irradiation increased not only production of momilactone B in rice seedlings but also secretion of momilactone B into rice rhizosphere. As momilactone B acts as an antimicrobial and allelopathic agent, secretion of momilactone B into the rhizosphere may provide a competitive advantage for root establishment through local suppression of soil microorganism and inhibition of the growth of competing plant species.

The relationship between coleoptile elongation and ethanolic fermentation was investigated in rice (Oryza sativa L.) coleoptiles of four cultivars subjected to a 48-h anoxic stress. The coleoptile elongation of all cultivars was suppressed by anoxic stress; however, the elongation of cvs Yukihikari and Nipponbare was much greater than that of cvs Leulikelash and Asahimochi. The stress did not significantly increase lactate dehydrogenase (LDH) activity or lactate concentration, but increased alcohol dehydrogenase (ADH) and pyruvate decarboxylase (PDC) activities, as well as ethanol concentration in the coleoptiles of all cultivars. The elevated ADH and PDC activities and ethanol concentration in cvs Yukihikari and Nipponbare were much greater than those of cvs Leulikelash and Asahimochi, suggesting that ethanolic fermentation is likely more active in cvs Yukihikari and Nipponbare than in cvs Leulikelash and Asahimochi. ATP concentration in cvs Yukihikari and Nipponbare in anoxia was also greater than that in cvs Leulikelash and Asahimochi in anoxia. The ethanol concentration in the coleoptiles was correlated with anoxia tolerance with respect to the ATP concentration and coleoptile elongation. These results suggest that the ability to increase ethanolic fermentation may be one of the determinants in anoxia tolerance of rice coleoptiles.

Germination of lettuce seeds was inhibited by 6-methoxy-2-benzoxazolinone (MBOA) at concentrations greater than 0.03 mmol/L. MBOA also inhibited the induction of alpha-amylase activity in the lettuce seeds at concentrations greater than 0.03 mmol/L. These two concentration-response curves for the germination and alpha-amylase indicate that the percentage of the germination was positively correlated with the activity of alpha-amylase in the seeds. Lettuce seeds germinated around 18h after incubation and inhibition of alpha-amylase by MBOA occurred within 6h after seed incubation. These results show that MBOA may inhibit the germination of lettuce seeds by inhibiting the induction of alpha-amylase activity.

Although most cereal roots cannot elongate under anoxic conditions, primary roots of three-day-old rice (Oryza sativa L.) seedlings were able to elongate during a 24-h period of anoxia. Hypoxic pretreatment (H-PT) increased the elongation of their roots. Sucrose synthase (EC 2.4.1.13), glucokinase (EC 2.7.1.2), fructokinase (EC 2.7.1.4), pyruvate decarboxylase (EC 4.1.1.1) and alcohol dehydrogenase (EC 1.1.1.1) activities were increased by anoxia in both H-PT and non-pretreated (N-PT) roots. However, these activities were greater in the H-PT roots than in the N-PT roots. The average rate of production of ethanol for the initial 6h after the onset of anoxia was 3.7 and 1.4 micromolg(-1) fresh weight h(-1) for the H-PT and N-PT roots, respectively, suggesting that ethanolic fermentation may increase more quickly in the H-PT roots than in the N-PT roots. Roots of the seedlings lost ATP and total adenine nucleotides in anoxia, however, the H-PT roots maintained higher levels of ATP and total adenine nucleotides compared to the N-PT roots. These results show that rice roots are able to utilize the set of enzymes involved in the metabolism of soluble sugars under anoxia. The ability to maintain an active fermentative metabolism for production of ATP by fueling the glycolytic pathway with fermentable carbohydrate is probably greater in H-PT than in N-PT roots.

Summary The changes in growth and gene expression of gibberellin (GA) 3β-hydroxylase were determined in dark-grown, 6-day-old, tall (cv. Alaska) and dwarf (cv. Progress No. 9) pea ( Pisum sativum L.) seedlings after transfer to conditions of red light irradiation. Red light inhibited the shoot elongation of Progress No. 9 much more so than that of Alaska, and the elongation of Progress No. 9 during 48 h was 23 percnt; that of Alaska. The transcript level of the GA 3β-hydroxylase gene was slightly greater in the shoots of dark-grown Alaska than that of dark-grown Progress No. 9. The red light decreased the transcript levels in both cultivars and the levels at 48 h were about 50 percnt; and 60 percnt; compared to the initial levels for Alaska and Progress No. 9, respectively. These results indicate that the difference in the transcript levels in both cultivars is not apparent. The only difference in GA 3β-hydroxylase proteins between Alaska and Progress No. 9 was the replacement of alanine with threonine at position 229 in their amino acid residues, which may affect the activity of the enzyme and may, at least partially, contribute to the growth habits of those cultivars. A possibility of additional function to distinguish the growth habits of those cultivars is also discussed here.

Summary Seedlings of Avena sativa were subjected to 24 h anoxic stress after 24 h hypoxic-pretreatment (H-PT). The H-PT increased 4.5-fold the survivability of the anoxia in coleoptiles of the seedlings compared to that of non-pretreated (N-PT) seedlings. During pretreatment periods, alcohol dehydrogenase (ADH, EC 1.1.1.1) and pyruvate decarboxylase (PDC, EC 4.1.1.1) activities in the coleoptiles became 4.1- and 3.9-fold greater respectively than those of N-PT seedlings. After the onset of anoxic stress, ADH and PDC activities in all coleoptiles increased, but their activities in the H-PT coleoptiles remained much greater than those in the N-PT coleoptiles, and the average production rate of ethanol for the initial 6 h was 3.1 and 1.4 μmol g −1 fresh weight h −1 for the H-PT and N-PT coleoptiles, respectively. Coleoptiles of the seedlings lost ATP rapidly under anoxic stress; however, the decrease in ATP was much slower in the H-PT coleoptiles than in the N-PT coleoptiles. These results suggest that the H-PT may improve the subsequent tolerance of anoxia in Avena coleoptiles due to maintenance of ATP levels with rapid induction of ethanolic fermentation. The present results are consistent with the results that were found in roots of several plants, suggesting that the mechanism in hypoxic acclimation to anoxia implicated in their roots may act in Avena coleoptiles.

The fern Gleichenia japonica often dominates plant communities by forming large monospecific stands throughout the temperate to tropical Asia. The objective of this study was the investigation of allelopathic property and substances of the decomposing litter of the fern to evaluate the possible involvement of its allelopathy in the domination. An aqueous methanol extract of G. japonica litter inhibited the growth of garden cress (Lepidium sativum), lettuce (Lactuca sativa), barnyard grass (Echinochloa crus-galli), and ryegrass (Lolium multiflorum). This result suggests that G. japonica litter contains growth inhibitory substances. The extract was purified by chromatography while monitoring the inhibitory activity, and a growth inhibitory substance was isolated. The chemical structure of the substance was determined by spectral data to be a novel compound, 13-O-β-fucopyranosyl-3β-hydroxymanool. This compound inhibited root and shoot growth of garden cress and barnyard grass at concentrations ranging from 89.7 to 271 μM for 50% inhibition. In addition, the compound had potent growth inhibitory activity with the soil taken from near the colony. The concentration of the compound in soil under a pure colony of G. japonica was 790 μM, suggesting that the compound may contribute to the establishment of monocultural stands by this fern.

Summary A new inhibitor of auxin-induced growth was isolated from light-grown shoots of maize seedlings (Zea mays L.) and its chemical structure was determined by spectral data as 5-chloro-6-methoxy-2-benzoxazolinone. It inhibited the auxin-induced growth of coleoptile sections of Avena seedlings, and the growth of shoots and roots of maize seedlings at concentrations higher than 1 and 3 and 10 gmol/L, respectively; the doses required for 20 % growth inhibition were 14.7, 15.8 and 292 gmol/L, respectively. The activity of the inhibitor was higher than that of its analogue, 6-methoxy-2-benzoxazolinone. These results suggest that a new growth inhibitor, 5-chloro-6-methoxy-2-benzoxazolinone, may be involved in the inhibition of auxin-induced growth, which may be one of the causes of inhibition of the growth of maize seedlings.

Summary Carrot (Daums carota L.) root tissues were stored under a continuous flow of 0.5 % or 2 % O2 (balance N2), or air at 5°C and 15°C for 7 days to determine the effects of low O2 atmosphere on lactic fermentation and to compare the relative importance of lactic fermentation with ethanolic fermentation. By day 2, lactate dehydrogenase (LDH, EC 1.1.1.27) activity and lactate concentration of samples in low O2 atmosphere was 1.6 to 2.8 and 2.6 to 5.6 times greater, respectively, than samples in air. The increase was greater at the lower O2 atmosphere and higher storage temperature. Alcohol dehydrogenase (ADH, EC 1.1.1.1) activity and ethanol concentration increased 2.4 to 8.2 and 11 to 31 times greater, respectively, than samples in air after 2 days of storage, with the increase being greater at the lower O2 atmosphere and higher storage temperature. Induced ADH was 8–12 times more active than LDH, and lactate accumulation was only 1 to 2% of ethanol accumulation in the carrots for a given treatment. Thus, although shredded carrot tissues exhibited LDH activity in a low O2 atmosphere, lactic fermentation appears to be a minor carbon flux compared to ethanolic fermentation.

Ginkgo ( Ginkgo biloba ) is one of the oldest living tree species and has been widely used in traditional medicine. Leaf extracts of ginkgo, such as the standardized extract EGb761, have become one of the best-selling herbal products. However, no bioactive compound directed at plants has been reported in this species. Therefore, we investigated possible allelopathic activity and searched for allelopathically active substances in ginkgo leaves. An aqueous methanol leaf extract inhibited the growth of roots and shoots of garden cress ( Lepidium sativum ), lettuce ( Lactuca sativa ), timothy ( Phleum pratense ) and ryegrass ( Lolium multiflorum ) seedlings. The extract was purified by several chromatographic runs and an allelopathically active substance was isolated and identified by spectral analysis to be the novel compound 2-hydroxy-6-(10-hydroxypentadec-11-enyl)benzoic acid. The compound inhibited root and shoot growth of garden cress and timothy at concentrations greater than 3 μM. The activity of the compound was 10- to 52-fold that of nonanoic acid. These results suggest that 2-hydroxy-6-(10-hydroxypentadec-11-enyl)benzoic acid may contribute to the allelopathic effect caused by ginkgo leaf extract. The compound may also have potential as a template for the development of new plant control substances.

The fern Gleichenia japonica is one of the most widely distributed fern and occurs throughout East to South Asia. The species often dominates plant communities by forming large monospecific colonies. However, the potential mechanism for this domination has not yet been described. The objective of this study was to test the hypothesis that allelochemicals are involved in the formation of G. japonica colonies. An aqueous methanol extract of G. japonica inhibited the growth of seedlings of garden cress (Lepidium sativum), lettuce (Lactuca sativa), ryegrass (Lolium multiflorum) and timothy (Phleum pratense). Increasing extract concentration increased the inhibition. These results suggest that G. japonica contain allelopathic substances. The extract was then purified by several chromatographies with monitoring the inhibitory activity and two growth inhibitory substances causing the allelopathic effect were isolated. The chemical structures of the two substances were determined by spectral data to be a novel compound 3-O-β-allopyranosyl-13-O-β-fucopyranosyl-3β-hydroxymanool (1) and 18-O-α- l -rhamnopyranosyl-(1→2)-β- d -glucopyranosyl-13-epitorreferol (2). These compounds inhibited the shoot and root growth of garden cress, lettuce, alfalfa (Medicago sativa), timothy, ryegrass and barnyardgrass (Echinochloa crus-galli) at concentrations greater than 0.1–1.0 mM. The concentrations required for 50% growth inhibition of root and shoot growth of these test plants ranged from 0.72 to 3.49 mM and 0.79 to 3.51 mM for compounds 1 and 2, respectively. Concentration of compounds 1 and 2 in soil under the pure colony of G. japonica was 4.9 and 5.7 mM, respectively, indicating concentrations over those required for 50% growth inhibition are potentially available under monocultural stands of these ferns. Therefore, these compounds may contribute to the allelopathic effects caused by presence of G. japonica and may thus contribute to the establishment of monocultural stands by this fern.

Although most allelochemicals can potentially cause growth inhibition in receiver plants, there is little information available about the absorption of these allelochemicals by the receiver plants. The present research describes the absorption of momilactone A and B by Arabidopsis thaliana (L.) and effects of the absorption on Arabidopsis growth. Exogenously applied momilactone A and B inhibited the growth of Arabidopsis hypocotyls and roots at concentrations greater than 10 and 1 μmol/L, respectively. The levels of momilactone A and B in Arabidopsis hypocotyls were approximately 3.2 and 2.4% of momilactone A and B, respectively, in the medium and those in Arabidopsis roots were about 3.9–3.4%, respectively. The absorption rates of momilactone A and B by Arabidopsis were not significantly different. The present research suggests that momilactone A and B may be absorbed in proportion to their applied levels, and the growth inhibitory effects of momilactone A and B may also correlated with their endogenous levels. However, the effectiveness of momilactone B on growth inhibition was much greater than that of momilactone A, and the sensitivities of hypocotyls to momilactone A and B were greater than those of roots. This is the first report describing the absorption of potent rice allelochemicals, momilactone A and B by receiver plants.

Here, we investigated chemical-mediated interaction between crop and weeds. Allelopathic activity of rice seedlings exhibited 5.3-6.3-fold increases when rice and barnyard grass seedlings were grown together, where there may be the competitive interference between rice and barnyard grass for nutrients. Barnyard grass is one of the most noxious weeds in rice cultivation. The momilactone B concentration in rice seedlings incubated with barnyard grass seedlings was 6.9-fold greater than that in rice seedlings incubated independently. Low nutrient growth conditions also increased allelopathic activity and momilactone B concentrations in rice seedlings. However, the increases in the low nutrient-induced allelopathic activity and momilactone B concentration were much lower than those in barnyard grass-induced allelopathic activity and momilactone B concentration. Root exudates of barnyard grass seedlings increased allelopathic activity and momilactone B concentration in rice seedlings at concentrations greater than 30 mg/L of the root exudates, and increasing the exudate concentration increased the activity and momilactone B concentration. Therefore, barnyard grass-induced allelopathic activity of rice seedlings may be caused not only by nutrient competition between two species, but also by components in barnyard grass root exudates. As momilactone B shows strong allelopathic activities, barnyard grass-induced allelopathic activity of rice may be due to the increased concentration of momilactone B in rice seedlings. The present research suggests that rice may respond to the presence of neighboring barnyard grass by sensing the components in barnyard grass root exudates and increasing allelopathic activity by production of elevated concentration of momilactone B. Thus, rice allelopathy may be one of the inducible defense mechanisms by chemical-mediated plant interaction between rice and barnyard grass, and the induced-allelopathy may provide a competitive advantage for rice through suppression of the growth of barnyard grass.

Physiological and metabolic responses to anoxia were compared for aerobically germinated seedlings of barley (Hordeum vulgare), oat (Avena sativa) and rice (Oryza sativa). Coleoptile growth of barley, oat and rice seedlings was suppressed by a 24 h-anoxic stress, but the growth of the rice coleoptiles was much greater than that of the barley and oat coleoptiles. ATP concentration in the anoxic rice coleoptiles was greater than that in the anoxic barley and oat coleoptiles. Concentrations of ethanol and activity of alcohol dehydrogenase (ADH) in the anoxic rice coleoptiles were also greater than those of the anoxic barley and oat coleoptiles, suggesting that ethanolic fermentation may be more active in the rice coleoptiles than in the barley and oat coleoptiles, where glycolysis and ethanolic fermentation are the main source of ATP production. Soluble sugar concentration in the anoxic rice coleoptiles was greater than that of the anoxic barley and oat coleoptiles. However, α-amylase, which catabolizes reserve starch to soluble sugars, was active in anoxic barley, oat and rice endosperms, and soluble sugar concentration in the anoxic barley, oat and rice endosperms was not significantly different. Therefore, anoxia stress may inhibit soluble sugar transport from the endosperms to the coleoptiles in barley and oat more than in rice. Since the availability of soluble sugar is essential for operation of glycolysis and fermentation in plant cells, ability for sugar transport from the endosperms to the coleoptiles may be one means to distinguish the coleoptile growth of these plant species in anoxia and anoxia tolerance of these plants.

Benzoxazinones and their degradation compounds inhibited root growth and α-amylase activity in cress seedlings. The inhibitory activity of these compounds was divided into three groups: the high active group; 2,4-dihydroxy-7-methoxy-(2 H )-1,4-benzoxazin-3(4 H )-one, 2,4-dihydroxy-(2 H )-1,4-benzoxazin-3(4 H )-one, 4-hydroxy-7-methoxy-(2 H )-1,4-benzoxazin-3(4 H )-one, 4-hydroxy-(2 H )-1,4-benzoxazin-3(4 H )-one, the moderate active group; 7-methoxy-(2 H )-1,4-benzoxazin-3(4 H )-one, (2 H )-1,4-benzoxazin-3(4 H )-one, 6-methoxy-benzoxazolin-2(3 H )-one, benzoxazolin-2(3 H )-one and 2-amino-phenoxazine-3-one, and the low active group; 2-hydroxy-(2 H )-1,4-benzoxazin-3(4 H )-one, 2-hydroxy-7-methoxy-(2 H )-1,4-benzoxazin-3(4 H )-one, 2-amino-7-hydroxyphenoxazine-3-one and 2-amino-7-methoxyphenoxazine-3-one. The structure–activity of these compounds suggests that compounds that have benzoxazinone skeletons are the most active structure, and a hydroxyl group at position C-2 on the benzoxazinone skeleton may not affect inhibitory activity, whereas a hydroxyl group at position N-4 on the skeleton is essential for inhibitory activity. However, the concentration–response curves of these compounds and the I 50 values (the concentrations required for 50% inhibition) for root growth and α-amylase indicated that root growth was positively correlated with the α-amylase activity in the seedlings. α-Amylase is required not only for seed germination, but also subsequent seedling growth until photosynthesis is sufficient to support seedling growth. Therefore, these results suggest that the compounds studied here may inhibit the root growth of cress seedlings by inhibiting α-amylase activity.

Summary In order to clarify the role of endogenous inhibitors of growth in dwarf and tall bean plants, a search for specific inhibitors of growth was undertaken using the neutral fractions of acetone extracts from the hypocotyls of dwarf (cv. Morocco) and tall (cv. Kentucky Wonder) bean seedlings ( Phaseolus vulgaris L. Three inhibitors were isolated from both cultivars and they were named N-1, N-2 and N-3, respectively, on the basis of the order of their elution from a silica-gel column. Variations in the activity of the inhibitors in hypocotyls of both cultivars after the onset of red-light irradiation were determined by use of a bean bioassay, and results were compared with those of red-light-induced inhibition of growth. The difference in the activity of N-1 between the two cultivars was particularly great, and the changes with time in levels of N-1 in hypocotyls of each cultivar matched the time course of the red-light-induced inhibition of growth of hypocotyls of the same cultivar. No such correlation was found with N-2 and N-3. These results suggest that red-light-induced inhibition of growth of bean hypocotyls may be controlled by variations in the levels of N-1. N-2 was spectroscopically identified as a mixture of xanthoxins.

Summary In order to correlate the role of endogenous inhibitors of growth of soybean plants with the inhibition of such growth by red light (R), a search for specific growth inhibitors was undertaken in the neutral and acidic fractions of acetone extracts from R- and dark-grown hypocotyls of soybean seedlings ( Glycine max L.). Two neutral and two acidic inhibitors were isolated. They were named N-I and N-II (neutral inhibitors) and A-I and A-II (acidic inhibitors), respectively, based on the order of their increasing Rf values during chromatography. Variations in the activity of the inhibitors in the hypocotyls of dark-grown seedlings after the onset of R irradiation were determined in terms of «cress units» which were defined as the activity that causes 50 % inhibition in the cress-root bioassay. The inhibitory activity of N-I and N-II tripled after exposure of plants to R for 24 h, whereas the activity of A-I and A-II remained almost unchanged. These results suggest that R-induced inhibition of growth of soybean hypocotyls may be controlled by variations in the levels of the neutral inhibitors, N-I and/or N-II, in the hypocotyls rather than by the acidic inhibitors.

Summary Effects of plant hormones on induction of alcohol dehydrogenase (ADH; EC 1.1.1.1) were determined. Exogenous auxin, abscisic acid (ABA) and cytokinin induced ADH in 6-day-old lettuce ( Lactuca sativa L.) seedlings, but gibberellin did not. The ADH activity was increased at concentrations greater than 1 μmol/L for auxin and ABA and 3 μmol/L for cytokinin, respectively, and all increases were found within 6 h after treatments. At 100 μmol/L doses, the activities in the seedlings were 3.9-, 3.2- and 1.9-fold greater than that of non-treated seedlings for auxin, ABA and cytokinin, respectively.

An aqueous methanol extract of the moss Rhynchostegium pallidifolium, which often forms large pure colonies on soils and rocks, inhibited the growth of cress (Lepidium sativum), alfalfa (Medicaga sativa), lettuce (Lepidium sativum), ryegrass (Lolium multiflorum), timothy (Phleum pratense) and Digitaria sanguinalis seedlings. Increasing the extract concentration increased the inhibition. These results suggest that R. pallidifolium may show allelopathic activity. The extract was purified and a putative compound causing this growth inhibitory effect was isolated. The chemical structure of the growth inhibitor was determined by MS, and (1)H and (13)C NMR spectral data as 3-hydroxy-beta-ionone. 3-Hydroxy-beta-ionone inhibited the shoot and root growth of cress seedlings at concentrations greater than 1 and 3 micromol/L, respectively. The doses required for 50% growth inhibition on the shoot and roots of cress seedlings were 16.3 and 14.9 micromol/L, respectively. The endogenous concentration of 3-hydroxy-beta-ionone in R. pallidifolium was 28.2 microg/g and the concentration of 3-hydroxy-beta-ionone in the growth medium of R. pallidifolium was 6.7 microg/g. These results suggest that 3-hydroxy-beta-ionone was likely secreted into the medium during the incubation of R. pallidifolium. In addition, 3-hydroxy-beta-ionone was found in the soil under the pure colonies of R. pallidifolium. Therefore, 3-hydroxy-beta-ionone may play an important role in the allelopathic activity of R. pallidifolium and may help competition with neighboring plants, resulting in the formation of pure colonies.

Nevertheless both plants are taxonomically quite distinct, momilactone A and B have been found only in rice and the moss, Hypnum plumaeforme which often forms large pure colonies. But biological meanings of momilactone A and B in H. plumaeforme is unknown. UV-irradiation induced a 15- and 16-fold increase in the secretion level of momilactone A and B, respectively, by H. plumaeforme into the growth medium. Jasmonic acid and the protein phosphatase inhibitor, cantharidin, also increased the momilactone A and B secretion levels by 12- to 15-fold. Cantharidin acts as an elicitor, jasmonic acid is an important signaling molecule regulating inducible defense genes against the pathogen infections. Therefore, elicitor and/or pathogen attacks may increase the secretion of momilactone A and B. As momilactone A and B are phytoalexic and allelopathic, the increasing secretion of momilactone A and B may be associated with the activation of the defense responses of H. plumaeforme in the rhizosphere where plants must compete with invading root systems of neighboring plants and prevent from bacteria and fungi infections. Momilactone A and B may be able to prevent H. plumaeforme from pathogen infections and help competition with neighboring plants resulting in the formation of pure colonies.

Aqueous methanol extracts of red pine (Pinus densiflora) needles inhibited the growth of roots and shoots of cress (Lepidium sativum), lettuce (Lactuca sativa), alfalfa (Medicago sativa), ryegrass (Lolium multiflorum), timothy (Pheleum pratense), Digitaria sanguinalis and Echinochloa crus-galli. Increasing the extract concentration increased inhibition, suggesting that the pine needles may have growth inhibitory substances and possess allelopathic potential. The aqueous methanol extract of the pine needles was purified, and a main inhibitory substance was isolated and determined by spectral data as 9alpha,13beta-epidioxyabeit-8(14)en-18-oic acid. This substance inhibited root and shoot growth of cress and Echinochloa crus-galli seedlings at concentrations greater than 0.1 mM. The endogenous concentration of the substance was 0.13 mmol/kg pine needle. These results suggest that 9alpha,13beta-epidioxyabeit-8(14)en-18-oic acid may contribute to the growth inhibitory effect of the pine needles and may play an important role in the allelopathy of red pine.

Exogenously applied ABA-beta-D-glucopyranosyl ester (ABA-GE) inhibited hypocotyl growth of Arabidopsis seedlings at concentrations greater than 0.3 micromol/L, and the concentration for 50% inhibition of hypocotyl growth was 1.8 micromol/L. ABA-beta-D-glucosidase activity in Arabidopsis seedlings was 17 nmol/mg protein/mim and increased by exogenously applied ABA-GE. The pH optimum of this enzyme in crude extract of Arabidopsis seedlings was 6.0 for the assay in the ABA-GE to ABA direction and its K(m) value for ABA-GE (pH 6.0) was 0.41 mmol/L. These results suggests that exogenously applied ABA-GE may be absorbed by roots and hydrolyzed by ABA-beta-D-glucosidase and librated free ABA may induce growth inhibition in Arabidopsis hypocotyls.

Rice seedlings (Oryza sativa L.) were incubated at 5-30 degrees C for 48 h and the effect of temperature on ethanolic fermentation in the seedlings was investigated in terms of low-temperature adaptation. Activities of alcohol dehydrogenase (ADH, EC 1.1.1.1) and pyruvate decarboxylase (PDC, EC 4.1.1.1) in roots and shoots of the seedlings were low at temperatures of 20-30 degrees C, whereas temperatures of 5, 7.5 and 10 degrees C significantly increased ADH and PDC activities in the roots and shoots. Temperatures of 5-10 degrees C also increased ethanol concentrations in the roots and shoots. The ethanol concentrations in the roots and shoots at 7.5 degrees C were 16- and 12-times greater than those in the roots and shoots at 25 degrees C, respectively. These results indicate that low temperatures (5-10 degrees C) induced ethanolic fermentation in the roots and shoots of the seedlings. Ethanol is known to prevent lipid degradation in plant membrane, and increased membrane-lipid fluidization. In addition, an ADH inhibitor, 4-methylpyrazole, decreased low-temperature tolerance in roots and shoots of rice seedlings and this reduction in the tolerance was recovered by exogenous applied ethanol. Therefore, production of ethanol by ethanolic fermentation may lead to low-temperature adaptation in rice plants by altering the physical properties of membrane lipids.

The release levels of a growth inhibitor, momilactone B, from rice (Oryza sativa L.) seedlings of eight cultivars were compared with the endogenous concentrations of momilactone B in their seedlings. All rice cultivars contained momilactone B in the seedlings, and their concentrations differed between the cultivars. Momilactone B was also found in all culture solutions in which these rice seedlings were grown, and the concentrations differed between the cultivars. The momilactone B concentrations in the culture solutions were reflected in the momilactone B concentrations in the seedlings. These results suggest that all rice cultivars may produce momilactone B and release momilactone B into the culture solutions. In addition, the release level of momilactone B may depend on the production level of momilactone B in the seedlings, which may affect allelopathic potential of these rice cultivars because as a growth inhibitor, momilactone B is able to act as an allelochemical.

Summary Since residues and extracts of rice plants were known to inhibit the germination and growth of several plant species, the possible involvement of a growth inhibitor, momilactone B, in rice allelopathy was discussed. Momilactone B was found in shoots and roots of rice plants over their entire life cycle. The level of momilactone B in shoots and roots increased with rice plant growing until flowering initiation, and then decreased. The highest level of momilactone B in the shoots and roots at the day of flowering initiation was 245 and 64.1 nmol g−1 fresh weight, respectively. Thus, 1 kg of rice shoots and roots, respectively, may be able to release 245 and 64.1 μmol of momilactone B into the soil or neighboring environment by decomposition of their residues, which may be sufficient to cause growth inhibition of their neighboring or successional plants. The growth inhibitory activity of momilactone B and the occurrence of momilactone B in rice plants suggest that momilactone B may contribute the growth inhibitory effect of rice residues and extracts, indicating that momilactone B may have an important role in the rice allelopathy.

Summary The correlation between changes in levels of the endogenous growth inhibitor, 3-hydroxy-β-ionone, and the growth recovery process after termination of 24-h irradiation with white light in dwarf bean seedlings ( Phaseolus vulgaris L. cv. Morocco) and tall bean seedlings (cv. Kentucky Wonder) was examined. The growth rate of hypocotyls of both cultivars increased, and the inhibitor in hypocotyls of both cultivars decreased, according to a sigmoidal time course with a half-life of 24 h. The relationship between concentration of the inhibitor of both cultivars and the corresponding growth rate is linear when plotted against the logarithms of the concentration of the inhibitor. The present results suggest that the endogenous growth inhibitor, 3-hydroxy-β-ionone, may play an important role in the growth recovery process of these cultivars after termination of light-irradiation.

Much research on rice allelopathy has been directed toward the selection of allelopathic rice strains and the identification of allelochemicals in rice. This paper briefly summarizes recent progress in the rice allelopathy and focuses on rediscovery of momilactone B as an allelochemical. A large number of rice varieties were found to inhibit the growth of several plant species when grown together under field and/or laboratory conditions. These findings suggest that rice probably produces and releases allelochemical(s) into the environment. The putative compound causing the inhibitory effect of rice was recently isolated from rice root exudates, and the chemical structure of the inhibitor was determined by spectral data as momilactone B. In addition, it has been found that momilactone B is released from rice roots into the neighboring environment, and the release level of momilactone B from rice may be sufficient to cause growth inhibition of neighboring plants. These findings suggest that momilactone B may play an important role in rice allelopathy.

Rice (Oryza sativa L.) seedlings were subjected to hypoxic pretreatment (H-PT; incubated in 5% O2 atmosphere) for various lengths of time followed by an anoxic stress. Anoxia tolerance of rice roots was improved with increasing duration of H-PT, but longer H-PT than 12 h gave no additional improvement. Concentrations of ATP and ethanol, and activities of pyruvate decarboxylase (EC 4.1.1.1) and alcohol dehydrogenase (EC 1.1.1.1) in the roots were increased by H-PT, and the times and patterns of increasing in these concentrations and activities were similar to those of increasing in the anoxia tolerance. These results suggest that the H-PT may increase anoxia tolerance due to maintenance of ATP levels with rapid induction of ethanolic fermentation, and hypoxic acclimation may occur within 12 h.

Summary Red light accelerated the elongation of the hooks of dark-grown soybean seedlings ( Glycine max L.) but suppressed the elongation of the elongation zone of the hypocotyls. The lag time prior to the acceleration of hook elongation by red light was longer by about 19 h than that associated with the suppression of elongation of the elongation zone. Twenty-seven h after the onset of red light, the enhanced elongation of the hook part balanced the reduced elongation of the elongation zone, and the plant resumed stern elongation at the maximum rate in spite of continued red irradiation. Subsequently, the growth rate of soybean seedlings exposed continuously to red light was equal to that of non-irradiated seedlings of the same age.