Syuntaro Hiradate

Detoxifying aluminium with buckwheat

Aluminium toxicity is a major problem limiting crop production in acid soils, which account for around 40% of the worlds arable land. Some plants have developed strategies to avoid or tolerate aluminium, including buckwheat (Fagopyrum esculentum Moench cv. Jianxi), which has a high resistance to aluminium, but the mechanism responsible for resistance is not known. We have found that the aluminium-resistant Juanxi cultivar of buckwheat secretes oxalic acid from its roots specifically and quickly in response to aluminium stress. Further, aluminium accumulates in the leaf cells in a non-toxic Al-oxalate complex with a 1:3 ratio of aluminium to oxalic acid.

Internal Detoxification Mechanism of Al in Hydrangea (Identification of Al Form in the Leaves).

An internal detoxification mechanism for Al was investigated in an Al-accumulating plant, hydrangea (Hydrangea macrophylla), focusing on Al forms present in the cells. The leaves of hydrangea contained as much as 15.7 mmol Al kg-1 fresh weight, and more than two-thirds of the Al was found in the cell sap. Using 27Al- nuclear magnetic resonance, the dominant peak of Al was observed at a chemical shift of 11 to 12 parts per million in both intact leaves and the extracted cell sap, which is in good accordance with the chemical shift for the 1:1 Al-citrate complex. Purification of cell sap by molecular sieve chromatography (Sephadex G-10) combined with ion-exclusion chromatography indicated that Al in fractions with the same retention time as citric acid contributed to the observed 27Al peak in the intact leaves. The molar ratio of Al to citric acid in the crude and purified cell sap approximated 1. The structure of the ligand chelated with Al was identified to be citric acid. Bioassay experiments showed that the purified Al complex from the cell sap did not inhibit root elongation of corn (Zea mays L.) and the viability of cells on the root tip surface was also not affected. These observations indicate that Al is bound to citric acid in the cells of hydrangea leaves.

Antimicrobial activity of culture filtrate of Bacillus amyloliquefaciens RC-2 isolated from mulberry leaves

ABSTRACT A potential antagonist, Bacillus amyloliquefaciens strain RC-2, against Colletotrichum dematium, mulberry anthracnose fungus, was obtained from healthy mulberry leaves by in vitro and in vivo screening techniques. Application of culture filtrate of RC-2 inhibited disease on mulberry leaves, indicating that suppression was due to antifungal compounds in the filtrate. Development of mulberry anthracnose on mulberry leaves was inhibited only when the culture filtrate was applied before fungal inoculation, and it was not inhibited by application after inoculation. These results suggest that the antifungal compounds in the filtrate exhibit a preventive effect on the disease. Peptone significantly increased production of the antifungal compounds. The culture filtrate of RC-2 also inhibited the growth of several other phytopathogenic fungi and bacteria, such as Rosellinia necatrix, Pyricularia oryzae, Agrobacterium tumefaciens, and Xanthomonas campestris pv. campestris, in vitro. From the culture filtrate of RC-2, seven kinds of antifungal compounds were isolated by high performance liquid chromatography analysis, and one of the compounds was determined as iturin A2, a cyclic peptide, by nuclear magnetic resonance and fast atom bombardment mass analysis.

Form of aluminium for uptake and translocation in buckwheat (Fagopyrum esculentum Moench).

Abstract. The forms of Al for uptake by the roots and translocation from the root to the shoot were investigated in a buckwheat (Fagopyrum esculentum Moench, cv. Jianxi) that accumulates Al in its leaves. The Al concentration in the xylem sap was 15-fold higher in the plants exposed to AlCl3 than in those exposed to an Al-oxalate (1:3) complex, suggesting that the roots take up Al in the ionic form. The Al concentration in the xylem sap was 4-fold higher than that in the external solution after a 1-h exposure to AlCl3 solution and 10-fold higher after a 2-h exposure. The Al concentration in the xylem sap increased with increasing Al concentration in the external solution. The Al uptake was not affected by a respiratory inhibitor, hydroxylamine, but significantly inhibited by the addition of La. These results suggest that Al uptake by the root is a passive process, and La3+ competes for the binding sites for Al3+ on the plasma membrane. The form of Al in the xylem sap was identified by 27Al-nuclear magnetic resonance analysis. The chemical shift of 27Al in the xylem sap was around 10.9 ppm, which is consistent with that of the Al-citrate complex. Furthermore, the dominant organic acid in the xylem sap was citric acid, indicating that Al was translocated in the form of Al-citrate complex. Because Al is present as Al-oxalate (1:3) in the root, the present data show that ligand exchange from oxalate to citrate occurs before Al is released to xylem.

Mulberry anthracnose antagonists (iturins) produced by Bacillus amyloliquefaciens RC-2

Bacillus amyloliquefaciens strain RC-2 produced seven antifungal compounds (1-7) secreted into the culture filtrate. These compounds inhibited the development of mulberry anthracnose caused by the fungus, Colletotrichum dematium. Chemical structural analyses by NMR and FAB-MS revealed that all these compounds were iturins (cyclic peptides with the following sequence: L-Asn --> D-Tyr --> D-Asn --> L-Gln --> L-Pro --> D-Asn --> L-Ser --> D-beta-amino acid -->) and compounds 1-6 are identical to iturins A-2-A-7, respectively. Compound 7 (iturin A-8) is a new iturin, which has a -(CH(2))(10)CH(CH(3))CH(2)CH(3) group as a side chain in the beta-amino acid in the molecule.

Allelopathic activity of buckwheat: isolation and characterization of phenolics

Abstract Laboratory and field experiments were conducted to assess the allelopathic potential of buckwheat. In the field, buckwheat demonstrated strong inhibitory activity by suppressing weeds. In laboratory studies, aqueous and organic solvent extracts of the aerial parts of common buckwheat inhibited the root and shoot growth of lettuce seedlings. The chloroform and ethyl acetate extracts showed maximum activity, and plants grown in the presence of the ethyl acetate extract showed severe root browning. The allelopathic constituents of the ethyl acetate phase were isolated and identified as gallic acid and (+)-catechin by nuclear magnetic resonance spectroscopy. Gallic acid and (+)-catechin were present in the upper part of buckwheat at concentrations of 0.02 and 0.01%, of fresh weight, respectively. Gallic acid was found to be selectively and strongly inhibitory to root and shoot growth of tested plants at 100 and 10 μg ml−1. (+)-Catechin, however, inhibited plant growth to a lesser extent. These results suggest that buckwheat may have allelopathic potential and that when used as a ground cover crop or green manure may produce inhibitors, which could suppress weeds. Nomenclature: Buckwheat, Fagopyrum esculentum Moench; lettuce, Lactuca sativa L.

First Isolation of Natural Cyanamide as a Possible Allelochemical from Hairy Vetch Vicia villosa

Cyanamide was isolated from the leaves and stems of hairy vetch (Vicia villosa), guided by plant growth inhibitory activity against lettuce (Lectuca sativa) seedlings. A large proportion of the inhibitory activity in the crude extract was explained by the presence of cyanamide, suggesting it to be a possible allelochemical in this species. The amount in a 9-day-old seedling, which had been grown without nutrients, reached approx. 40 times that of a nongerminated seed, demonstrating cyanamide biosynthesis in the seedlings. This is the first report on the isolation of a possible allelochemical from hairy vetch and also of the finding of cyanamide as a natural product.

Carbon source of humic substances in some Japanese volcanic ash soils determined by carbon stable isotopic ratio, δ13C

Abstract Volcanic ash soils (including Andisols) have been known to accumulate extremely large amounts of humic acids in their thick surface horizons, and their accumulation mechanism is of interest. Two mechanisms have been proposed: (1) active metals like Al and Fe, supplied from weathered volcanic materials, stabilize humic acids through complexation reactions; (2) cultivated Japanese pampas grasses ( Miscanthus sinensis A.) and its charred materials are the major carbon source of humic acids. In the present study, contribution ratio of the pampas grass (C4-plant) on the carbons of the humic acids was determined by measuring their stable isotopic ratio of carbon (δ 13 C). In Japanese volcanic ash soils, humic acids were originated from both C3- and C4-plants, and the contribution ratio of C4-plants (mostly pampas grass) ranged from 18% to 52%. Highly humified (dark-colored) humic acids tended to show higher contribution ratio of C4-plants among volcanic ash soils, although the major part of the carbon had originated from C3-plants. It was also clarified that the δ 13 C values of crude soil samples correlated well with those of humic and fulvic acids. Therefore, reported δ 13 C values of crude soil samples in the literature would be useful for estimating the carbon source of soil humic substances. Literature survey of the δ 13 C values of crude soil samples also indicated that a large part of the carbon in humic substances has originated from C3-plants rather than C4-plants, implying the importance of the active metals (such as Al and Fe) on the formation and accumulation of the humic acids in volcanic ash soils.

Chemical species of Al reacting with soil humic acids.

The formation and presence of aluminum tridecamer (Al(13), [AlO(4)Al(12)(OH)(24)(H(2)O)(12)](7+)) in solution was strongly inhibited by soil humic acids (HA), as shown by liquid-state 27Al-nuclear magnetic resonance (NMR) analyses. It was also observed that Al(13) coprecipitated with HA in the form of Al(13)-HA complexes, as shown by solid-state magic angle spinning (MAS) 27Al-NMR analyses. Tetrahedral Al in the Al(13)-HA complexes was gradually converted to octahedral Al, indicating that Al(13) in Al(13)-HA complexes was decomposed into Al-HA complexes with reaction time. When HA was present in the acidic Al solution before the partial neutralization, the precipitates formed after the partial neutralization did not contain any tetrahedral Al, indicating the absence of Al(13) and that HA is a strong inhibitor of Al(13) formation. These results indicate that Al(13) is not formed from Al complexed with soil HA after partial neutralization. If Al(13) was added to soils, it would preferentially form precipitates of non-phytotoxic Al(13)-HA complex in the presence of excess HA (i.e. a COOH/Al(13) molar ratio of more than 9) and gradually be converted to an octahedral Al-HA complex. Therefore, Al(13) is not a major plant-growth inhibitor in humus-rich acidic soils.

Pyrolysis Temperature-Dependent Changes in Dissolved Phosphorus Speciation of Plant and Manure Biochars

Pyrolysis of plant and animal wastes produces a complex mixture of phosphorus species in amorphous, semicrystalline, and crystalline inorganic phases, organic (char) components, and within organo-mineral complexes. To understand the solubility of different phosphorus species, plant (cottonseed hull) and manure (broiler litter) wastes were pyrolyzed at 350, 500, 650, and 800 °C and exposed to increasingly more rigorous extraction procedures: water (16 h), Mehlich 3 (1 mM EDTA at pH 2.5 for 5 min), oxalate (200 mM oxalate at pH 3.5 for 4 h), NaOH-EDTA (250 mM NaOH + 5 mM EDTA for 16 h), and total by microwave digestion (concentrated HNO3/HCl + 30% H2O2). Relative to the total (microwave digestible) P, the percentage of extractable P increased in the following order: M3 < oxalate ≈ water < NaOH-EDTA for plant biochars and water < M3 < NaOH-EDTA < oxalate for manure biochars. Solution phase (31)P NMR analysis of NaOH-EDTA extracts showed the conversion of phytate to inorganic P by pyrolysis of manure and plant wastes at 350 °C. Inorganic orthophosphate (PO4(3-)) became the sole species of ≥ 500 °C manure biochars, whereas pyrophosphate (P2O7(4-)) persisted in plant biochars up to 650 °C. These observations suggested the predominance of (i) amorphous (rather than crystalline) calcium phosphate in manure biochars, especially at ≥ 650 °C, and (ii) strongly complexed pyrophosphate in plant biochars (especially at 350-500 °C). Correlation (Pearsons) was observed (i) between electric conductivity and ash content of biochars with the amount of inorganic P species and (ii) between total organic carbon and volatile matter contents with the organic P species.