Sequential loss-on-ignition as a simple method for evaluating the stability of soil organic matter under actual environmental conditions.

Soil organic matter (SOM) is one of the largest carbon (C) reservoirs on Earth, and therefore its stability attracts a great deal of interest from the perspective of the global C cycle. This study examined the applicability of loss-on-ignition with a stepwise increase in temperature (SIT-LOI) of soil to evaluate the stability of SOM using soil samples having different organic matter (OM) and mineral contents and different mean residence times (MRTs) for SOM. The responses of SOM to the SIT-LOI varied depending on the samples but were all successfully approximated by a liner regression model as a function of the temperature of LOI. The slope value in the liner model that determines the residual potential of carbon during the SIT-LOI highly correlated with MRT of SOM, suggesting that this value reflects the overall stability of SOM over a range of soil properties. This hypothesis was consistent with the observation that Δ14C values of SOM decreased with increasing LOI temperature and thus, older, slower-cycling SOM was preferentially left in the soil samples by SIT-LOI. Additionally, the hypothesis was also supported by the significant correlations (p < 0.01) between the slope value and OM and mineral contents in the samples because these components are considered to regulate SOM stability. In addition to the regression analysis of the SIT-LOI data, changes in carbon to nitrogen (C/N) and carbon to hydrogen (C/H) ratios and stable carbon isotope signatures (δ13C) of the samples were investigated. The results suggest that the mineral association of SOM is an important factor characterizing the response of SOM to LOI. Hence, it was concluded that SIT-LOI is a simple and useful method for evaluating the stability of SOM under actual environmental conditions.

The (oxalato)aluminate complex as an antimicrobial substance protecting the "shiro" of Tricholoma matsutake from soil micro-organisms.

Tricholoma matsutake, a basidiomycete, forms ectomycorrhizas with Pinus densiflora as the host tree. Its fruiting body, "matsutake" in Japanese, is an edible and highly prized mushroom, and it grows in a circle called a fairy ring. Beneath the fairy ring of T. matsutake, a whitish mycelium-soil aggregated zone, called "shiro" in Japanese, develops. The front of the shiro, an active mycorrhizal zone, functions to gather nutrients from the soil and roots to nourish the fairy ring. Bacteria and sporulating fungi decrease from the shiro front, whereas they increase inside and outside the shiro front. Ohara demonstrated that the shiro front exhibited antimicrobial activity, but the antimicrobial substance has remained unidentified for 50 years. We have identified the antimicrobial substance as the (oxalato)aluminate complex, known as a reaction product of oxalic acid and aluminum phosphate to release soluble phosphorus. The complex protects the shiro from micro-organisms, and contributes to its development.

Soil phosphorous is the primary factor determining species-specific plant growth depending on soil acidity in island ecosystems with severe erosion.

Disturbances caused by invasive ungulates alter soil environments markedly and can prevent ecosystem recovery even after eradication of the ungulates. On oceanic islands, overgrazing and trampling by feral goats has caused vegetation degradation and soil erosion, which can alter soil chemistry. To understand the effects of the changes on plant performance, we conducted a laboratory experiment to assess herbaceous species growth under various soil conditions with phosphorous, nutrients, and acidity. Subsoil was collected from Nakodo-jima in the northwest Pacific. Six herbaceous species dominating the island were grown in soils with three levels of added CaCO3 and P2O5 and two levels of added KNO3. After 4 weeks of growth, the total dry plant weight was significantly lower with no added P2O5, regardless of the addition of KNO3. Three species weighed more under P2O5 and KNO3 addition in high-pH soil, whereas the remaining three weighed less. Our results indicated that herbaceous species growth is limited primarily by phosphorous availability; the limitation is dependent on soil pH, and the trend of dependency differs among species. This implies that ecosystems with extreme disturbances cannot recover without improving the soil chemistry.

Predicting ecosystem changes by a new model of ecosystem evolution.

In recent years, computer simulation has been increasingly used to predict changes in actual ecosystems. In these studies, snapshots of ecosystems at certain points in time were instantly constructed without considering their evolutionary histories. However, it may not be possible to correctly predict future events unless their evolutionary processes are considered. In this study, we developed a new ecosystem model for reproducing the evolutionary process on an oceanic island, targeting Nakoudojima Island of the Ogasawara Islands. This model successfully reproduced the primitive ecosystem (the entire island covered with forest) prior to the invasion of alien species. Also, by adding multiple alien species to this ecosystem, we were able to reproduce temporal changes in the ecosystem of Nakoudojima Island after invasion of alien species. Then, we performed simulations in which feral goats were eradicated, as had actually been done on the island; these suggested that after the eradication of feral goats, forests were unlikely to be restored. In the ecosystems in which forests were not restored, arboreous plants with a high growth rate colonized during the early stage of evolution. As arboreous plants with a high growth rate consume a large amount of nutrient in soil, creating an oligotrophic state. As a result, plants cannot grow, and animal species that rely on plants cannot maintain their biomass. Consequently, many animals and plants become extinct as they cannot endure disturbances by alien species, and the ecosystem loses its resilience. Therefore, even if feral goats are eradicated, forests are not restored. Thus, the founder effect from the distant past influences future ecosystem changes. Our findings show that it is useful to consider the evolutionary process of an ecosystem in predicting its future events.

Overcoming the difficulties in collecting apoplastic fluid from rice leaves by the infiltration-centrifugation method.

Physiological and biochemical studies on the leaf apoplast have been facilitated by the use of the infiltration-centrifugation technique to collect intercellular washing fluid (IWF). However, this technique has been difficult to implement in rice (Oryza sativa L.) for various reasons. We compared the collection efficiency of leaf IWF between two types of rice varieties (Indica and Japonica), as well as between rice and other species (spinach, snap bean and wheat). Although the extraction of IWF in most species took only 2-3 min, it took up to 35 min in rice. The difficulty in infiltration with rice was ascribed to the small stomatal aperture and hydrophobicity of the leaves. In this study, we have established an improved method for collecting IWF and determining the apoplastic air and water volumes in rice leaves. We have shortened the infiltration time to 8 min via the following improvements: (i) infiltration under outdoor shade in the daytime to prevent stomatal closure and a rise in temperature of the infiltration medium; (ii) soaking of leaves in a surfactant solution to decrease the leaf hydrophobicity; and (iii) continuous pressurization using a sealant injector to facilitate the infiltration. The rapid collection of IWF achieved using this technique will facilitate study of the leaf apoplast in rice.

A new class of mealybug pheromones: a hemiterpene ester in the sex pheromone of Crisicoccus matsumotoi.

Mealybugs, which include several agricultural pests, are small sap feeders covered with a powdery wax. They exhibit clear sexual dimorphism; males are winged but fragile and short lived, whereas females are windless and less mobile. Thus, sex pheromones emitted by females facilitate copulation and reproduction by serving as a key navigation tool for males. Although the structures of the hitherto known mealybug pheromones vary among species, they have a common structural motif; they are carboxylic esters of monoterpene alcohols with irregular non-head-to-tail linkages. However, in the present study, we isolated from the Matsumoto mealybug, Crisicoccus matsumotoi (Siraiwa), a pheromone with a completely different structure. Using gas chromatography-mass spectrometry and nuclear magnetic resonance spectroscopy, we identified the pheromone as 3-methyl-3-butenyl 5-methylhexanoate. Its attractiveness to males was confirmed in a series of field trapping experiments involving comparison between the isolated natural product and a synthetic sample. This is the first report of a hemiterpene mealybug pheromone. In addition, the acid moiety (5-methylhexanoate) appears to be rare in insect pheromones.

Quantification of cyanamide in young seedlings of Vicia species, Lens culinaris, and Robinia pseudo-acacia by gas chromatography-mass spectrometry.

We quantified the cyanamide content of young leaves of nine Vicia species, Lens culinaris, and Robinia pseudo-acacia using a modified analytical procedure that made it possible to measure the cyanamide content of a single leaf. Recent molecular phylogenetic analysis suggests that cyanamide is present in V. benghalensis, which is placed in a monophyletic group with cyanamide-biosynthesizing plants, V. villosa and V. cracca; this suggestion was verified.

A single application of fertilizer can affect semi-natural grassland vegetation over half a century.

Restoration of species-rich semi-natural grassland requires not only a seed source but also appropriate soil properties. In Europe, approximately 10 years are required for the properties of fertilized soils to reach suitable conditions and be considered successfully restored. However, restoration may require additional time in Japan because heavier precipitation causes leaching of basic cations from soils, resulting in soil acidification; volcanic ejecta also forms active Al and Fe hydroxides with high phosphate sorption. Within this context, we aimed to answer the following questions: i) whether and how the impacts of fertilization remain in the soil properties after half a century in Japan; and ii) how fertilization affects the restoration of semi-natural grasslands in Japan. We investigated the vegetation and soil properties of a Zoysia japonica pasture improved half a century ago with a single application of fertilizer and an adjacent semi-natural grassland (native pasture) in Japan, and found the following: (1) the two pastures had similar dominance of Z. japonica, but differed in the species composition; (2) the improved pasture exhibited lower species richness than the native pasture; (3) soil nutrients, including N, P, K, Mg, and Ca, were higher in the improved pasture than in the native pasture; and (4) many chemical properties of the soils were associated with species composition; namely, the vegetation on nutrient-rich soil had more alien species and fewer native species. We conclude that a single dose of fertilization can affect soil properties in semi-natural grasslands over half a century in Japan, leading to species loss and changing the species composition. We suggest that fertilized soils under grazing in Japan may require more than half a century to restore the nutrients to suitable levels for the establishment of a species-diverse grassland.

Microarray analysis of Arabidopsis plants in response to allelochemical L-DOPA.

Velvetbean (Mucuna pruriens) plants impede the growth of neighboring plants. One compound, 3-(3',4'-dihydroxyphenyl)-L-alanine (L-DOPA), is responsible for the allelopathic capacity of velvetbean. This compound is an active allelochemical that decreases root growth of several plant species. In mammals, L-DOPA is a well-known therapeutic agent for the symptomatic relief of Parkinson's disease. However, its mode of action in plants is still not well understood. To address such issues, gene expression in Arabidopsis thaliana plants, which had been exposed to L-DOPA, was analyzed using DNA microarrays. After 6 h of L-DOPA exposure, the expression of 110 genes was significantly upregulated, and the expression of 69 genes was significantly downregulated. These induced genes can be divided into different functional categories, mainly on the basis of subcellular localization, metabolism, and proteins with a binding function or cofactor requirement. Based on these results, we suggest that L-DOPA acts by two mechanisms: it influences amino acid metabolism and deregulates metal homeostasis, especially that of iron, which is required for the fundamental biological processes of all organisms.

Nocardioides sp. strain WSN05-2, isolated from a wheat field, degrades deoxynivalenol, producing the novel intermediate 3-epi-deoxynivalenol.

The mycotoxin deoxynivalenol (DON) causes serious problems worldwide in the production of crops such as wheat and barley because of its toxicity toward humans and livestock. A bacterial culture capable of degrading DON was obtained from soil samples collected in wheat fields using an enrichment culture procedure. The isolated bacterium, designated strain WSN05-2, completely removed 1,000 µg/mL of DON from the culture medium after incubation for 10 days. On the basis of phylogenetic studies, WSN05-2 was classified as a bacterium belonging to the genus Nocardioides. WSN05-2 showed significant growth in culture medium with DON as the sole carbon source. High-performance liquid chromatography analysis indicated the presence of a major initial metabolite of DON in the culture supernatant. The metabolite was identified as 3-epi-deoxynivalenol (3-epi-DON) by mass spectrometry and (1)H and (13)C nuclear magnetic resonance analysis. The amount of DON on wheat grain was reduced by about 90% at 7 days after inoculation with WSN05-2. This is the first report of a Nocardioides sp. strain able to degrade DON and of the yet unknown 3-epi-DON as an intermediate in the degradation of DON by a microorganism.

Bleaching of leaf litter accelerates the decomposition of recalcitrant components and mobilization of nitrogen in a subtropical forest.

Selective removal of lignin and other recalcitrant compounds, collectively registered as acid-unhyrolyzable residue (AUR), results in bleaching of leaf litter, but the importance of bleaching in decomposition processes on forest soil has not been fully evaluated. The aims of this study were to elucidate the occurrence of bleached area in decomposing leaf litter and to compare chemical composition between bleached and nonbleached portions in a subtropical forest in Japan. Field incubation of leaf litter was performed over an 18-month period with the litterbag method. The decomposition processes during the first 9 month were characterized by the relatively rapid mass loss and increase of bleached area, whereas the mass loss was slowed down and the bleached area decreased thereafter. Mass loss of leaf tissues was faster and AUR content was lower in bleached than in nonbleached portions, indicating the acceleration of mass loss in bleached leaf tissues by the selective decomposition of recalcitrant compounds. The decrease in carbonyl-C in the bleached portions was associated with the increase of extractable nitrogen. The results suggest that the bleaching plays a dominant role in the transformation and turnover of organic compounds and nitrogen in decomposing leaf litter.

Ionomic Responses of Local Plant Species to Natural Edaphic Mineral Variations.

Leaf ionome indicates plant phylogenetic evolution and responses to environmental stress, which is a critical influential factor to the structure of species populations in local edaphic sites. However, little is known about leaf ionomic responses of local plant species to natural edaphic mineral variations. In the present study, all plant species and soil samples from a total of 80 soil sites in Shiozuka Highland were collected for multi-elemental analysis. Ioniomic data of species were used for statistical analysis, representing 24 species and 10 families. Specific preferences to ionomic accumulation in plants were obviously affected by the phylogeny, whereas edaphic impacts were also strong but limited within the phylogenetic preset. Correlations among elements resulted from not only elemental synergy and competition but also the adaptive evolution to withstand environmental stresses. Furthermore, ionomic differences of plant families were mainly derived from non-essential elements. The majority of variations in leaf ionome is undoubtedly regulated by evolutionary factors, but externalities, especially environmental stresses also have an important regulating function for landscape formation, determining that the contributions of each factor to ionomic variations of plant species for adaptation to environmental stress provides a new insight for further research on ionomic responses of ecological speciation to environmental perturbations and their corresponding adaptive evolutions.

Characterization of root mucilage from Melastoma malabathricum, with emphasis on its roles in aluminum accumulation.

Plant roots exude viscous polysaccharides, called mucilage. One of the suggested roles of mucilage is immobilization of toxic metal cations, including aluminum (Al), in the rhizosphere. Mucilage exuded from roots of Melastoma malabathricum (Al accumulator) was characterized in comparison with that of Zea mays (maize; Al nonaccumulator). Removal of mucilage significantly reduced Al accumulation in M. malabathricum. The cation adsorption affinity of M. malabathricum mucilage was higher for Al and lanthanum (La) than for barium (Ba), whereas that of maize mucilage was in the order Ba > La > Al. A (27)Al nuclear magnetic resonance (NMR) spectrum of the Al-adsorbed mucilage and bioassay with alfalfa seedlings indicated that the concentrated Al in the mucilage of M. malabathricum, unlike that of maize, bound very weakly to cation exchange sites of mucilage. The higher charge density in M. malabathricum mucilage, derived from unmethylated uronic acid, is inferred to be related to preferential adsorption of trivalent cation. Not only a higher degree of methylation in the uronic acid (glucuronic acid) but also H(+) release from roots to the mucilage appears to be responsible for the loose binding of Al in M. malabathricum mucilage. These characteristics of mucilage may help Al hyperaccumulation in M. malabathricum.

Mechanism for the detoxification of aluminum in roots of tea plant (Camellia sinensis (L.) Kuntze).

To determine the mechanism of aluminum (Al) detoxification in the roots of tea plants (Camellia sinensis (L.) Kuntze), the amounts of Al and Al-chelating compounds (fluoride (F), organic acids and catechins) were measured and the chemical forms of Al in root cell extracts were identified by the application of 27Al-nuclear magnetic resonance (NMR) spectroscopy. Tea plants were cultivated in nutrient solutions containing 0, 4, 1.0 and 4.0 mM of Al at pH 4.2 for approximately 10 weeks. The levels of soluble Al, water-soluble oxalate and citrate, but not F, malate or catechins in young roots increased with an increase in the concentration of Al in the treatment solution. The 27Al NMR spectra of root tips and cell sap extracted from root tips that had been treated with Al were almost identical and had four signals, with two (11 and 16 ppm) apparently corresponding to the known chemical shifts of Al-oxalate complexes. In the spectra of cell sap, the resonances at 11 and 16 ppm increased with an increase in the Al contents. These results suggest that the levels of Al-oxalate complexes increased in response to an increase in the Al level, implying that oxalate is a key Al-chelating compound in the mechanism of Al detoxification in the tea root.

Limited distribution of natural cyanamide in higher plants: occurrence in Vicia villosa subsp. varia, V. cracca, and Robinia pseudo-acacia.

Cyanamide (NH2CN) has recently been proven to be a natural product, although it has been synthesized for over 100 years for agricultural and industrial purposes. The distribution of natural cyanamide appears to be limited, as indicated by our previous investigation of 101 weed species. In the present study, to investigate the distribution of natural cyanamide in Vicia species, we monitored the cyanamide contents in V. villosa subsp. varia, V. cracca, and V. amoena during their pre-flowering and flowering seasons. It was confirmed that V. cracca was superior to V. villosa subsp. varia in accumulating natural cyanamide, and that V. amoena was unable to biosynthesize this compound under laboratory condition examined. The localization of cyanamide in the leaves of V. villosa subsp. varia seedlings was also clarified. In a screening study to find cyanamide-biosynthesizing plants, only Robinia pseudo-acacia was found to contain cyanamide among 452 species of higher plants. We have investigated 553 species to date, but have so far found the ability to biosynthesize cyanamide in only three species, V. villosa subsp. varia, V. cracca and R. pseudo-acacia.

Adsorption of herbicidally active degradate 2-(2,4-dichloro-3-methylphenoxy)propanoic acid on an andosol.

The adsorption of 2-(2,4-dichloro-3-methylphenoxy)propanoic acid (DMPA) on the surface horizon of a humus-rich Andosol was examined. To investigate the mechanisms of adsorption, chemically treated Andosols, such as organic matter removed Andosol, organic matter and active metals removed Andosol, and clay minerals of the Andosol, were prepared. Furthermore, humic acid was extracted from the Andosol. The mechanisms of the DMPA adsorption were identified by using those untreated and chemically treated Andosols and the humic acid. The amount of DMPA adsorbed increased with decreasing equilibrium pH value. Active surface hydroxyl groups were identified as the most important soil functional group in DMPA adsorption. The predominant mechanism of DMPA adsorption on the Andosol is a ligand-exchange reaction, in which an active surface hydroxyl on Al and/or Fe is replaced by a carboxylic group of DMPA. A comparative study revealed that the amount of DMPA adsorbed was slightly greater than that of (2,4-dichlorophenoxy)acetic acid (2,4-D), especially at equilibrium pH values below 5. This is because the octanol-water partition coefficient (log Kow) of DMPA in the equilibrium pH range is higher than that of 2,4-D, and SOM participates in the adsorption process through a hydrophobic interaction.

Adsorption of 2,4-dichlorophenoxyacetic acid by an Andosol.

To identify the important soil components involved in 2,4-dichlorophenoxyacetic acid (2,4-D) adsorption on Andosols, 2,4-D adsorption on a surface horizon of an Andosol was compared with that on hydrogen peroxide (H2O2)-treated (soil organic matter [SOM] was removed), acid-oxalate (OX)-treated (active metal hydroxides and SOM were removed), and dithionite-citrate-bicarbonate (DCB)-treated (free and active metal [hydr]oxides and SOM were removed) soil samples at equilibrium pHs ranging from 4 to 8. Although the untreated soil contained a large amount of organic C (71.9 g kg-1), removal of SOM had little effect on 2,4-D adsorption. Active surface hydroxyls, which were attached to the active and free metal (hydr)oxides and metal SOM complexes, were identified as the most important soil functional group for 2,4-D adsorption. The dominant mechanism of the 2,4-D adsorption was a ligand exchange reaction in which the carboxylic group of 2,4-D displaced the active surface hydroxyl associated with metals and formed a strong coordination bond between the 2,4-D molecule and soil solid phase. The ligand exchange reaction reasonably accounted for the selective adsorption of 2,4-D over Cl-, competitive adsorption of phosphate over 2,4-D, reduction in plant-growth-inhibitory activity of soil-adsorbed 2,4-D, and the high 2,4-D adsorption ability of Andosols. Although a humic acid purified from the soil did not adsorb 2,4-D, the presence of the humic acid increased 2,4-D adsorption on Al and Fe, probably by inhibiting the hydrolysis and polymerization of Al and Fe resulting in the preservation of available adsorption sites on these metals. The adsorption behavior of 2,4-D on soils could be a good index for predicting the adsorption behavior of other organic acids in soils.

Biodegradation of methylthio-s-triazines by Rhodococcus sp. strain FJ1117YT, and production of the corresponding methylsulfinyl, methylsulfonyl and hydroxy analogues.

A novel bacterial strain FJ1117YT was isolated from an enrichment culture with the herbicide simetryn. The isolate was capable of degrading the herbicide supplied as the sole sulfur source in an aquatic batch culture. The strain FJ1117YT was identified as that belonging to Rhodococcus sp. on the basis of comparative morphology, physiological characteristics and comparison of the 16S rRNA gene sequence. The biodegradation pathway of simetryn was established by isolating the methylsulfinyl analogue as the first metabolite and by identification of the methylsulfonyl intermediate and the hydroxy analogue by liquid chromatography-mass spectrometry (LC-MS) and/or nuclear magnetic resonance (NMR) analysis. The results indicate that the methylthio group was progressively oxidised and hydrolysed by the strain FJ1117YT. The same strain is also able to metabolise other methylthio-s-triazines such as ametryn, desmetryn, dimethametryn and prometryn through similar pathways.

Phyllosphere Methylobacterium bacteria contain UVA-absorbing compounds.

Microbes inhabiting the phyllosphere encounter harmful ultraviolet rays, and must develop adaptive strategies against this irradiation. In this study, we screened bacterial isolates originating from the phyllosphere of various plants which harbored absorbers of ultraviolet A (UVA), a wavelength range which is recognized as harmful to human skin. Of the 200 phyllosphere bacterial isolates we screened, methanol extracts from bacterial cells of seventeen isolates absorbed wavelengths in the range of 315-400nm. All of the UVA-absorbing strains belonged to Methylobacterium species based on 16S ribosomal RNA gene sequences, suggesting that cells of this bacterial genus contain specific UVA-absorbing compounds. When cells of a representative Methylobacterium strain were extracted using various solvents, UVA absorption was observed in the extracts obtained using several aqueous solvents, indicating that the UVA-absorbing compounds were highly polar. A compound was purified using solid columns and HPLC separation, and comparative analysis revealed that the absorption strength and spectrum of the compound were similar to those of the known UVA filter, avobenzone. The compound was also verified to be stable under UVA exposure for at least 480min. Based on these results, the UVA-absorbing compound harbored by Methylobacterium has potential to be used as a novel sunscreen ingredient.

Evidence of cyanamide production in hairy vetch Vicia villosa.

Cyanamide (NH(2)CN) has recently been isolated as a plant growth inhibitor from Vicia villosa, which is the first discovery of cyanamide from natural sources. To reveal the presence of the biosynthesized cyanamide in plants, 3.4 mM potassium ((15)N)nitrate was administered to 15- to 35-day-old plants of V. villosa, from which the cyanamide was purified and subjected to GC/MS analysis. The isotopic ratio (15)N/((14)N + (15)N) of the cyanamide was calculated to be 0.143, while that of the cyanamide extracted from V. villosa grown in the presence of a natural N source was 0.0065. The (15)N-enrichment proved de novo biosynthesis of cyanamide.