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.

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.

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.

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.

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.

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.

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.

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.

Contribution of soil esterase to biodegradation of aliphatic polyester agricultural mulch film in cultivated soils.

The relationship between degradation speed of soil-buried biodegradable polyester film in a farmland and the characteristics of the predominant polyester-degrading soil microorganisms and enzymes were investigated to determine the BP-degrading ability of cultivated soils through characterization of the basal microbial activities and their transition in soils during BP film degradation. Degradation of poly(butylene succinate-co-adipate) (PBSA) film was evaluated in soil samples from different cultivated fields in Japan for 4 weeks. Both the degradation speed of the PBSA film and the esterase activity were found to be correlated with the ratio of colonies that produced clear zone on fungal minimum medium-agarose plate with emulsified PBSA to the total number colonies counted. Time-dependent change in viable counts of the PBSA-degrading fungi and esterase activities were monitored in soils where buried films showed the most and the least degree of degradation. During the degradation of PBSA film, the viable counts of the PBSA-degrading fungi and the esterase activities in soils, which adhered to the PBSA film, increased with time. The soil, where the film was degraded the fastest, recorded large PBSA-degrading fungal population and showed high esterase activity compared with the other soil samples throughout the incubation period. Meanwhile, esterase activity and viable counts of PBSA-degrading fungi were found to be stable in soils without PBSA film. These results suggest that the higher the distribution ratio of native PBSA-degrading fungi in the soil, the faster the film degradation is. This could be due to the rapid accumulation of secreted esterases in these 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 (Pearson's) 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.

Design and synthesis of conformationally constrained analogues of cis-cinnamic acid and evaluation of their plant growth inhibitory activity.

1-O-cis-Cinnamoyl-ß-D-glucopyranose is known to be one of the most potent allelochemical candidates and was isolated from Spiraea thunbergii Sieb by Hiradate et al. (2004), who suggested that it derived its strong inhibitory activity from cis-cinnamic acid, which is crucial for phytotoxicity. In this study, key structural features and substituent effects of cis-cinnamic acid (cis-CA) on lettuce root growth inhibition was investigated. These structure-activity relationship studies indicated the importance of the spatial relationship of the aromatic ring and carboxylic acid moieties. In this context, conformationally constrained cis-CA analogues, in which the aromatic ring and cis-olefin were connected by a carbon bridge, were designed, synthesized, and evaluated as plant growth inhibitors. The results of the present study demonstrated that the inhibitory activities of the five-membered and six-membered bridged compounds were enhanced, up to 0.27 µM, and were ten times higher than cis-CA, while the potency of the other compounds was reduced.

Substituent effects of cis-cinnamic acid analogues as plant growh inhibitors.

1-O-cis-Cinnamoyl-ß-D-glucopyranose is one of the most potent allelochemicals that has been isolated from Spiraea thunbergii Sieb by Hiradate et al. It derives its strong inhibitory activity from cis-cinnamic acid (cis-CA), which is crucial for phytotoxicity. By preparing and assaying a series of cis-CA analogues, it was previously found that the key features of cis-CA for lettuce root growth inhibition are a phenyl ring, cis-configuration of the alkene moiety, and carboxylic acid. On the basis of a structure-activity relationship study, the substituent effects on the aromatic ring of cis-CA were examined by systematic synthesis and the lettuce root growth inhibition assay of a series of cis-CA analogues having substituents on the aromatic ring. While ortho- and para-substituted analogues exhibited low potency in most cases, meta-substitution was not critical for potency, and analogues having a hydrophobic and sterically small substituent were more likely to be potent. Finally, several cis-CA analogues were found to be more potent root growth inhibitors than cis-CA.

Effect of aluminum on metabolism of organic acids and chemical forms of aluminum in root tips of Eucalyptus camaldulensis Dehnh.

Eucalyptus (Eucalyptus camaldulensis) has relatively high resistance to aluminum (Al) toxicity than the various herbaceous plants and model plant species. To investigate Al-tolerance mechanism, the metabolism of organic acids and the chemical forms of Al in the target site (root tips) in Eucalyptus was investigated. To do this, 2-year old rooted cuttings of E. camaldulensis were cultivated in half-strength Hoagland solution (pH 4.0) containing Al (0, 0.25, 0.5, 1.0, 2.5 and 5.0mM) salts for 5weeks; growth was not affected at concentrations up to 2.5mM even with Al concentration reaching 6000µgg(-1) DW. In roots, the citrate content also increased with increasing Al application. Concurrently, the activities of aconitase and NADP(+)-isocitrate dehydrogenase, which catalyze the decomposition of citrate, decreased. On the other hand, the activity of citrate synthase was not affected at concentrations up to 2.5mM Al. (27)Al-NMR spectroscopic analyses were carried out where it was found that Al-citrate complexes were a major chemical form present in cell sap of root tips. These findings suggested that E. camaldulensis detoxifies Al by forming Al-citrate complexes, and that this is achieved through Al-induced citrate accumulation in root tips via suppression of the citrate decomposition pathway.

Linking temperature sensitivity of soil organic matter decomposition to its molecular structure, accessibility, and microbial physiology.

Temperature sensitivity of soil organic matter (SOM) decomposition may have a significant impact on global warming. Enzyme-kinetic hypothesis suggests that decomposition of low-quality substrate (recalcitrant molecular structure) requires higher activation energy and thus has greater temperature sensitivity than that of high-quality, labile substrate. Supporting evidence, however, relies largely on indirect indices of substrate quality. Furthermore, the enzyme-substrate reactions that drive decomposition may be regulated by microbial physiology and/or constrained by protective effects of soil mineral matrix. We thus tested the kinetic hypothesis by directly assessing the carbon molecular structure of low-density fraction (LF) which represents readily accessible, mineral-free SOM pool. Using five mineral soil samples of contrasting SOM concentrations, we conducted 30-days incubations (15, 25, and 35 °C) to measure microbial respiration and quantified easily soluble C as well as microbial biomass C pools before and after the incubations. Carbon structure of LFs (<1.6 and 1.6-1.8 g cm(-3) ) and bulk soil was measured by solid-state (13) C-NMR. Decomposition Q10 was significantly correlated with the abundance of aromatic plus alkyl-C relative to O-alkyl-C groups in LFs but not in bulk soil fraction or with the indirect C quality indices based on microbial respiration or biomass. The warming did not significantly change the concentration of biomass C or the three types of soluble C despite two- to three-fold increase in respiration. Thus, enhanced microbial maintenance respiration (reduced C-use efficiency) especially in the soils rich in recalcitrant LF might lead to the apparent equilibrium between SOM solubilization and microbial C uptake. Our results showed physical fractionation coupled with direct assessment of molecular structure as an effective approach and supported the enzyme-kinetic interpretation of widely observed C quality-temperature relationship for short-term decomposition. Factors controlling long-term decomposition Q10 are more complex due to protective effect of mineral matrix and thus remain as a central question.

Plant growth inhibitor from the Malaysian medicinal plant Goniothalamus andersonii and related species.

A crude methanol extract of Goniothalamus andersonii J. Sinclair strongly inhibited elongation of lettuce (Lactuca sativa L.) radicles. We conducted bioassay-guided purification of G. andersonii bark extract and obtained goniothalamin as the major bioactive compound. Its EC50 values against elongation of lettuce radicles and hypocotyls were 50 and 125 micromol L(-1), respectively. Among the six species tested, timothy was the most sensitive to goniothalamin. Quantification of this compound in other Goniothalamus species suggested that the plant inhibitory activity of this genus is explainable by goniothalamin, with G. calcareus as an exception.

Key structural features of cis-cinnamic acid as an allelochemical.

1-O-cis-cinnamoyl-ß-D-glucopyranose is one of the most potent allelochemicals isolated from Spiraea thunbergii Sieb. It is suggested that it derives its strong inhibitory activity from cis-cinnamic acid, which is crucial for phytotoxicity. It was synthesized to confirm its structure and bioactivity, and also a series of cis-cinnamic acid analogues were prepared to elucidate the key features of cis-cinnamic acid for lettuce root growth inhibition. The cis-cyclopropyl analogue showed potent inhibitory activity while the saturated and alkyne analogues proved to be inactive, demonstrating the importance of the cis-double bond. Moreover, the aromatic ring could not be replaced with a saturated ring. However, the 1,3-dienylcyclohexene analogue showed strong activity. These results suggest that the geometry of the C-C double bond between the carboxyl group and the aromatic ring is essential for potent inhibitory activity. In addition, using several light sources, the photostability of the cinnamic acid derivatives and the role of the C-C double bond were also investigated.

Degradation of biodegradable plastic mulch films in soil environment by phylloplane fungi isolated from gramineous plants.

To improve the biodegradation of biodegradable plastic (BP) mulch films, 1227 fungal strains were isolated from plant surface (phylloplane) and evaluated for BP-degrading ability. Among them, B47-9 a strain isolated from the leaf surface of barley showed the strongest ability to degrade poly-(butylene succinate-co-butylene adipate) (PBSA) and poly-(butylene succinate) (PBS) films. The strain grew on the surface of soil-mounted BP films, produced breaks along the direction of hyphal growth indicated that it secreted a BP-degrading enzyme, and has directly contributing to accelerating the degradation of film. Treatment with the culture filtrate decomposed 91.2 wt%, 23.7 wt%, and 14.6 wt% of PBSA, PBS, and commercially available BP polymer blended mulch film, respectively, on unsterlized soil within 6 days. The PCR-DGGE analysis of the transition of soil microbial community during film degradation revealed that the process was accompanied with drastic changes in the population of soil fungi and Acantamoeba spp., as well as the growth of inoculated strain B47-9. It has a potential for application in the development of an effective method for accelerating degradation of used plastics under actual field conditions.

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.