Suppressive effect of the deep placement of lime nitrogen on N2O emissions in a soybean field.

Deep placement of slow-release nitrogen (N) fertilizers improves the growth and yield of soybean with a high N use efficiency. This study examined the effectiveness of deep placement of lime nitrogen (LN) in reducing N2O emissions in a soybean field and compared it with conventional fertilization. Before sowing soybeans, the starter N fertilizer (16 kg-N ha-1 ammonium sulfate) was mixed in the surface soil and the following four treatments were installed: the control with only the starter N (CT), conventional top-dressing of 60 kg-N ha-1 coated urea (CV), deep placement (20 cm depth) of 100 kg-N ha-1 urea (DU), and deep placement (20 cm depth) of 100 kg-N ha-1 LN (DL). The seasonal patterns of N2O emission rates measured using the closed chamber method differed among the treatments: in CT, N2O emissions were relatively low; in CV, N2O emissions derived from the top-dressed coated urea were observed from 91 days after sowing; in DU and DL, deeply-placed N was converted to N2O in the early growth stages. The cumulative N2O emissions in DL (1.8 kg-N ha-1) during the soybean cultivation period were significantly lower than those in DU (3.1 kg-N ha-1) and CV (2.8 kg-N ha-1), and slightly higher than CT (1.2 kg-N ha-1). The magnitude of N2O emissions was significantly lower in DL than DU, indicating that the choice of N fertilizer is important to reduce N2O emissions. Focusing on N2O emissions per unit coarse grain yield of soybeans, the value in DL was 0.45 g-N kg-1, which was significantly lower than 0.74 g-N kg-1 in CV. In conclusion, the deep placement of LN has the potential to be a sustainable farming method that can promote yields and reduce N2O emissions in soybean cultivation for high yield with N fertilization.

Multiplexed ISSR genotyping by sequencing distinguishes two precious coral species (Anthozoa: Octocorallia: Coralliidae) that share a mitochondrial haplotype.

BACKGROUND: Precious corals known as coralliid corals (Anthozoa: Octocorallia) play an important role in increasing the biodiversity of the deep sea. Currently, these corals are highly threatened because of overfishing that has been brought on by an increased demand and elevated prices for them.The deep sea precious corals Pleurocorallium elatius and P. konojoi are distributed in Japanese waters and have distinct morphological features: (1) the terminal branches of the colony form of P. elatius are very fine, while those of P. konojoi are blunt and rounded, (2) the autozooids of P. elatius are arranged in approximately four rows, while those of P. konojoi are clustered in groups. However, previous genetic analysis using mtDNA and nuclear DNA did not indicate monophyly. Therefore, it is important to clarify their species status to allow for their conservation. METHODOLOGY: We collected a total of 87 samples (60 of Corallium japonicum and 27 of P. konojoi) from around the Ryukyu Islands and Shikoku Island, which are geographically separated by approximately 1,300 km. We used a multiplexed inter-simple sequence repeat (ISSR) genotyping by sequencing (MIG-seq) and obtained 223 SNPs with which to perform STRUCTURE analysis and principle coordinate analysis (PCoA). In addition, two relatively polymorphic mtDNA regions were sequenced and compared. RESULTS: P. elatius and P. konojoi share a same mtDNA haplotype, which has been previously reported. However, MIG-seq analysis clearly distinguished the two species based on PCoA and STRUCTURE analysis, including 5% of species-specific fixed SNPs. CONCLUSION: This study indicated that P. elatius and P. konojoi are different species and therefore both species should be conserved separately. Our findings highlight the importance of the conservation of these two species, especially P. elatius, whose population has been dramatically depleted over the last 100 years. The study also demonstrated the effectiveness and robustness of MIG-seq for defining closely related octocoral species that were otherwise indistinguishable using traditional genetic markers (mtDNA and EF).

Differences in Soil Bacterial Community Compositions in Paddy Fields under Organic and Conventional Farming Conditions.

Soil bacterial community compositions and temporal changes in organic paddy fields were elucidated using a 16S rRNA gene amplicon sequencing analysis with a high-throughput next generation sequencer. At transplanting, bacterial community compositions in organic and conventional paddy fields were mostly similar despite differences in field management. The bacterial community composition in organic fields differed from that under conventional management during the rice growth period, possibly as a result of the decomposition process of organic fertilizers. However, differences in the frequency of tillage and photosynthetic bacterial inoculations in the organic plots had less of an impact on bacterial communities.

Measurement and estimation of radiocesium discharge rate from paddy field during land preparation and mid-summer drainage.

In this research, we evaluated the range of (137)Cs discharge rates from paddy fields during land preparation and mid-summer drainage. First, we investigated (137)Cs discharge loads during land preparation and mid-summer drainage and their ratio to the (137)Cs inventory of paddy field soil. We found that total discharge rates were 0.003-0.028% during land preparation and 0.001-0.011% during mid-summer drainage. Next, we validated the range of obtained total discharge of (137)Cs from the paddy fields using a simplified equation and literature review. As a result, we conclude that the range of total outflow loads of suspended solids for the investigated paddy field was generally representative of paddy fields in Japan. Moreover, the (137)Cs discharge ratio had a wide range, but was extremely small relative to (137)Cs present in paddy field soil before irrigation.

Radiocesium immobilization to leaf litter by fungi during first-year decomposition in a deciduous forest in Fukushima.

Vast forest areas in eastern Japan have been contaminated with radio-isotopes by the Fukushima Daiichi Nuclear Power Plant (FDNPP) accident. Radiocesium (radioCs) is known to remain bioavailable in forest ecosystems for a long time, and it is necessary to terminate the cycling process to decontaminate the forest ecosystem. We observed radiocesium concentrations of leaf litter during decomposition on a forest floor where radiocesium ((137)Cs) contamination was ∼155 kBq/m(2). Litter bag experiments were conducted with newly fallen mixed deciduous leaf litter in a deciduous forest (alt. 610 m) about 50 km from the FDNPP. Litter bags were retrieved in April, June, August, October, and December 2012. Fresh litter (137)Cs concentration was ∼3000 Bq/kg in December 2011. During the decomposition process on the forest floor, litter (137)Cs concentration increased rapidly and exceeded 25,000 Bq/kg after 6 months, whereas potassium (K) concentration in the litter was rather stable, indicating that radiocesium and K showed contrasting dynamics during the early decomposition phase. Nitrogen, phosphorus, and (137)Cs contents were positively correlated to fungal biomass, evaluated by phospholipid fatty acids in the litter during decomposition. The increase of radiocesium concentration mainly occurred during from April to October, when fungal growth peaked. Therefore, this suggests fungal translocation of nutrients from outside the litter substrate (immobilization) is the mechanism to increase radiocesium in the decomposing litter. The amount of (137)Cs contained in the 1-year-old decomposed leaf litter was estimated to be 4% per area of the soil-contaminated (137)Cs.

4,4'-DDE and Endosulfan Levels in Agricultural Soils of the Çukurova Region, Mediterranean Turkey.

Mediterranean Turkey has long been at the forefront of Turkish agriculture and the use of organochlorinated pesticides (OCPs) in this area rose considerably between the 1940s and 1980s. This study aimed to determine OCP residue levels in agricultural soils collected from the Mersin and Adana Districts, Çukurova Basin in Mediterranean Turkey. Most soil samples were contaminated with one, or both, of two OCP metabolites; 4,4'-dichlorodiphenyldichloroethylene (4,4'-DDE) and endosulfan sulfate. 4,4'-DDE occurred in 27 of the 29 samples and ranged from 6 to 1090 µg kg(-1)-dry soil (ds)(-1), while six samples contained endosulfan sulfate ranging between 82 and 1226 µg kg(-1)-ds(-1). Generally, horticultural and corn-planted soils contained only 4,4'-DDE, whereas greenhouse cultivation appeared to accumulate both residues. This study indicated that 4,4'-DDE occurred above acceptable levels of risk in agricultural soils of Mersin District and further studies on the qualitative and quantitative assessment of OCPs in other agricultural regions with intensive pesticide use are necessary to fully understand the impact of OCPs on agricultural soil in Turkey.

Sulfate-reducing bacteria mediate thionation of diphenylarsinic acid under anaerobic conditions.

Diphenylarsinic acid (DPAA) is often found as a toxic intermediate metabolite of diphenylchloroarsine or diphenylcyanoarsine that were produced as chemical warfare agents and were buried in soil after the World Wars. In our previous study Guan et al. (J Hazard Mater 241-242:355-362, 2012), after application of sulfate and carbon sources, anaerobic transformation of DPAA in soil was enhanced with the production of diphenylthioarsinic acid (DPTAA) as a main metabolite. This study aimed to isolate and characterize anaerobic soil microorganisms responsible for the metabolism of DPAA. First, we obtained four microbial consortia capable of transforming DPAA to DPTAA at a high transformation rate of more than 80% after 4 weeks of incubation. Sequencing for the bacterial 16S rRNA gene clone libraries constructed from the consortia revealed that all the positive consortia contained Desulfotomaculum acetoxidans species. In contrast, the absence of dissimilatory sulfite reductase gene (dsrAB) which is unique to sulfate-reducing bacteria was confirmed in the negative consortia showing no DPAA reduction. Finally, strain DEA14 showing transformation of DPAA to DPTAA was isolated from one of the positive consortia. The isolate was assigned to D. acetoxidans based on the partial 16S rDNA sequence analysis. Thionation of DPAA was also carried out in a pure culture of a known sulfate-reducing bacterial strain, Desulfovibrio aerotolerans JCM 12613(T). These facts indicate that sulfate-reducing bacteria are microorganisms responsible for the transformation of DPAA to DPTAA under anaerobic conditions.

Characterization of the proteinaceous skeletal organic matrix from the precious coral Corallium konojoi.

The Japanese red and pink corals are known to be precious because of their commercial value resulting from their use in ornaments, jewelry, and medicine. Precious corals are very interesting models for biomineralization studies and possess two different skeletal structures: an axial skeleton and an endoskeleton (sclerites). Although it has long been known that the organic matrix proteins existing in coral skeletons are critical for the oriented precipitation of CaCO3 crystals, these proteins in moderate deep-sea Japanese precious corals remain uncharacterized. Therefore, in this study, we performed skeletal whole proteome analyses using 1D and 2D electrophoresis, nano-LC, and MALDI-TOF-TOF MS. We identified a total of 147 functional coral skeletal organic matrix proteins (120 from the sclerites and 36 from the axial skeleton), including two calcium-binding calmodulin. Among the organic matrix proteins identified, nine key proteins are highly typical and common in both skeletons. Strong glycosylation activity, which is essential for skeletal formation in calcifying organisms, was detected in both skeletons. This work demonstrates unique biomineralization-related proteins in precious corals and provides the first description of the major proteinaceous components of CaCO3 minerals in precious corals, enabling the comparative investigation of biocalcification in other octocorals.

¹³7Cs in irrigation water and its effect on paddy fields in Japan after the Fukushima nuclear accident.

There is concern that radiocesium deposited in the environment after the accident at the Fukushima Daiichi Nuclear Power Plant (FDNPP) in March 2011 will migrate to paddy fields through hydrological pathways and cause serious and long-lasting damage to the agricultural activities. This study was conducted in the Towa region of Nihonmatsu in the northern part of Fukushima Prefecture, Japan, (1) to quantify (137)Cs in stream water used to irrigate paddy fields by separating the dissolved and particulate components in water samples and then fractionating the particulate components bonded in different ways using a sequential extraction procedure, and (2) to determine the amounts of radiocesium newly added to paddy fields in irrigation water relative to the amounts of radiocesium already present in the fields from the deposition of atmospheric fallout immediately after the FDNPP accident. Three catchments were studied, and the (137)Cs activity concentrations in stream water samples were 79-198 mBq L(-1) under stable runoff conditions and 702-13,400 Bq L(-1) under storm runoff conditions. The residual fraction (F4, considered to be non-bioavailable) was dominant, accounting for 59.5-82.6% of the total (137)Cs activity under stable runoff conditions and 69.4-95.1% under storm runoff conditions. The (137)Cs newly added to paddy fields in irrigation water only contributed 0.03-0.05% of the amount already present in the soil (201-348 kBq m(-2)). This indicates that the (137)Cs inflow load in irrigation water is negligible compared with that already in the soil. However, the contribution from the potentially bioavailable fractions (F1+F2+F3) was one order of magnitude larger, accounting for 0.20-0.59%. The increase in the dissolved and soluble radiocesium fraction (F1) was especially large (3.0% to infinity), suggesting that radiocesium migration in irrigation water is increasing the accumulation of radiocesium in rice.

Purification and characterization of a novel fibrinolytic enzyme from culture supernatant of Pleurotus ostreatus.

A fibrinolytic enzyme was produced by an edible mushroom of Pleurotus ostreatus using submerged culture fermentation. The enzyme was purified from the culture supernatant by applying a combination of freeze-thaw treatment, ammonium sulfate precipitation, hydrophobic interaction, and gel filtration chromatographies. The enzyme was purified by a 147-fold, with a yield of 7.54%. The molecular masses of the enzyme an determined by gel filtration and SDSPAGE were 13.6 and 18.2 kDa, respectively. The isoelectric point of the enzyme was 8.52. It hydrolyzed fibrinogen by cleaving the α and ß chains of fibrinogen followed by the γ chains, and also activated plasminogen into plasmin. The enzyme was optimally active at 45°C and pH 7.4. The enzyme activity was completely inhibited by EDTA, whereas protease inhibitors of TPCK, SBTI, PMSF, aprotinin and pepstatin showed no inhibition on its activity. The partial amino acid sequences of the enzyme as determined by Q-TOF2 were ATFVGCSATR, GGTLIHESSHFTR, and YTTWFGTFVTSR. These sequences showed a high degree of homology with those of metallo-endopeptidases from P. ostreatus and Armillaria mellea. The purified enzyme can also be applied as a natural agent for oral fibrinolytic therapy or prevention of thrombosis.

Formation of diphenylthioarsinic acid from diphenylarsinic acid under anaerobic sulfate-reducing soil conditions.

Diphenylarsinic acid (DPAA) is a toxic phenylarsenical compound often found around sites contaminated with phenylarsenic chemical warfare agents, diphenylcyanoarsine or diphenylchloroarsine, which were buried in soil after the World Wars. This research concerns the elucidation of the chemical structure of an arsenic metabolite transformed from DPAA under anaerobic sulfate-reducing soil conditions. In LC/ICP-MS analysis, the retention time of the metabolite was identical to that of a major phenylarsenical compound synthesized by chemical reaction of DPAA and hydrogen sulfide. Moreover the mass spectra for the two compounds measured using LC/TOF-MS were similar. Subsequent high resolution mass spectral analysis indicated that two major ions at m/z 261 and 279, observed on both mass spectra, were attributable to C12H10AsS and C12H12AsSO, respectively. These findings strongly suggest that the latter ion is the molecular-related ion ([M+H](+)) of diphenylthioarsinic acid (DPTA; (C6H5)2AsS(OH)) and the former ion is its dehydrated fragment. Thus, our results reveal that DPAA can be transformed to DPTA, as a major metabolite, under sulfate-reducing soil conditions. Moreover, formation of diphenyldithioarsinic acid and subsequent dimerization were predicted by the chemical reaction analysis of DPAA with hydrogen sulfide. This is the first report to elucidate the occurrence of DPAA-thionation in an anaerobic soil.

Enhanced transformation of diphenylarsinic acid in soil under sulfate-reducing conditions.

Diphenylarsinic acid (DPAA) is known to be the major contaminant in soils where diphenylchloroarsine and diphenylcyanoarsine were abandoned after World Wars I and II. In this study, experimental model studies were performed to elucidate key factors regulating the transformation of DPAA under anaerobic soil conditions. The elimination of DPAA in Gleysol soils (Qiqihar and Shindori soils) was more rapid than in Mollisol and Regosol soils (Heihe and Ikarashi soils, respectively) during a 5-week incubation. No clear relationship between decreasing rates of DPAA concentrations and soil Eh values was found. The Ikarashi soil showed the slowest decrease in DPAA concentrations among the four soils, but the transformation of DPAA was notably enhanced by addition of exogenous sulfate together with acetate, cellulose or rice straw. Addition of molybdate, a specific inhibitor of sulfate reduction, resulted in the stagnation of DPAA transformation, suggesting that indigenous sulfate reducers play a role in DPAA transformation under anaerobic conditions. Arsenate, phenylarsonic acid, phenylmethylarsinic acid, diphenylmethylarsine oxide and three unknown compounds were detected as metabolites of DPAA. This is the first study to reveal enhancement of DPAA transformation under sulfate-reducing conditions.

Soil radiocesium distribution in rice fields disturbed by farming process after the Fukushima Dai-ichi Nuclear Power Plant accident.

A magnitude 9.0 earthquake and subsequent large tsunami hit the northeastern coast of Japan on March 11, 2011. This resulted in serious damage to the reactors of the Fukushima Dai-ichi Nuclear Power Plant (FDNPP), operated by the Tokyo Electric Power Company. Large amounts of radionuclides were released from the FDNPP, a proportion of which were deposited onto the ground. In this study, we investigated soil radiocesium contamination of rice fields in Aga and Minamiuonuma, Niigata, ~130 and 200 km away from the FDNPP, respectively, as Niigata is one of the largest rice growing regions in Japan. Soil samples were collected from the plow layer of five rice fields in August and September, 5-6 months after the FDNPP accident. Results showed that radiocesium concentrations (the sum of Cs-134 and Cs-137) in the rice soil samples were ~300 Bq (kg dry soil)(-1). All samples contained a Cs-134/Cs-137 activity ratio of 0.68-0.96 after correction to March 11, 2011, showing that the radiocesium released from the FDNPP were deposited on these areas. Although the rice fields had been disturbed by farming processes after the FDNPP accident, the depth distribution of radiocesium concentrations in the plow layers showed higher concentrations in the upper soil layers. This suggests that spring tillage, flooding and puddling performed before rice transplantation may not disperse radiocesium deposited on the surface through the whole plow layer. In addition, the planar distribution of radiocesium concentrations was examined near the water inlet in one of the rice fields. Highest activities were found aligned with the direction of irrigation water discharge, indicating that radioactivity levels in rice fields may be elevated by an influx of additional radionuclides, probably in irrigation water, during farming.

The Melithaeidae (Cnidaria: Octocorallia) of the Ryukyu Archipelago: molecular and morphological examinations.

The family Melithaeidae (Octocorallia: Alcyonacea) is distributed in the West Pacific, Indian Ocean and the Red Sea. They are most abundant in warmer waters but can also be found in temperate waters. At present six genera are assigned to this family (Melithaea, Mopsella, Clathraria, Acabaria, Wrightella and Asperaxis), however overlapping characteristics make this group's taxonomic identification difficult and their relationships unclear. There are only a few reports from the Ryukyu Archipelago in southern Japan of melithaeids and most other octocorals, despite the islands being an area of high octocoral diversity. To help resolve the taxonomic confusion in this family, samples from various Ryukyu Archipelago locations were collected and DNA sequences of nuclear 28S ribosomal DNA and mitochondrial cytochrome oxidase I (COI) were obtained. Additionally, SEM micrographs of the sclerites of specimens were taken to further confirm the molecular results. Three strongly supported clades were recovered from the COI and 28S rDNA analyses, corresponding to Melithaea, Acabaria, and Mopsella, and in most cases clades were clearly related with the sclerite shape reported for each genus. These results show clearly that molecular differences are present between the three genera, and also demonstrates the strong need of other molecular markers for resolving intra-generic phylogenies. Our results provide baseline data for future studies of this octocoral family, not only on taxonomy, but also with regards to their distribution in the Ryukyu Islands.

Host-related variability in arbuscular mycorrhizal fungal structures in roots of Hedera rhombea, Rubus parvifolius, and Rosa multiflora under controlled conditions.

The arbuscular mycorrhizal (AM) morphology of three host plant species inoculated with single and mixed fungal culture and the distribution of AM fungal species in roots of the hosts treated with a mixed culture of AM fungi were determined. The aim was to investigate the effect of host plants and AM fungi on AM morphology of coexisting plant species. Noncolonized rooted cuttings of Hedera rhombea (Miq) Bean, Rubus parvifolius L., and Rosa multiflora Thunb. were inoculated with five fungal species as single and mixed culture inocula. The fungal species used were Gigaspora rosea and Scutellospora erythropa, previously isolated from H. rhombea; Acaulospora longula and Glomus etunicatum from R. parvifolius; and Glomus claroideum from both plant species. A few hyphal and arbusculate coils were seen in the mixed culture-inoculated roots of R. parvifolius; all fungal treatments produced this Paris-type AM in H. rhombea and Arum-type AM in R. parvifolius, and R. multiflora indicates that AM morphology is strongly controlled by the identity of the host plants used in this study. AM fungal rDNA was extracted separately from roots of each replicate plant species inoculated with the mixed fungal culture, amplified, cloned, sequenced, and analyzed to determine the AM fungal species and their respective proportions in roots of each plant species. Glomus etunicatum and G. claroideum of the family Glomaceae generally occurred more frequently in R. parvifolius and R. multiflora, which form Arum-types, whereas S. erythropa, of the family Gigasporaceae, was the most frequently detected species in H. rhombea, which produced Paris-type AM. Although the genotype of the plant species used appears to determine the AM morphologies formed, there was preferential association between the hosts and AM fungal inoculants.

Cooccurring plants forming distinct arbuscular mycorrhizal morphologies harbor similar AM fungal species.

Arbuscular mycorrhizal (AM) fungal spores were isolated from field transplants and rhizosphere soil of Hedera rhombea (Miq) Bean and Rubus parvifolius L., which form Paris-type and Arum-type AM, respectively. DNA from the spore isolates was used to generate molecular markers based on partial large subunit (LSU) ribosomal RNA (rDNA) sequences to determine AM fungi colonizing field-collected roots of the two plant species. Species that were isolated as spores and identified morphologically and molecularly were Gigaspora rosea and Scutellospora erythropa from H. rhombea, Acaulospora longula and Glomus etunicatum from R. parvifolius, and Glomus claroideum from both plants. The composition of the AM fungal communities with respect to plant trap cultures was highly divergent between plant species. Analysis of partial LSU rDNA sequences amplified from field-collected roots of the two plant species with PCR primers designed for the AM fungi indicated that both plants were colonized by G. claroideum, G. etunicatum, A. longula, and S. erythropa. G. rosea was not detected in the field-collected roots of either plant species. Four other AM fungal genotypes, which were not isolated as spores in trap cultures from the two plant species, were also found in the roots of both plant species; two were closely related to Glomus intraradices and Glomus clarum. One genotype, which was most closely related to Glomus microaggregatum, was confined to R. parvifolius, whereas an uncultured Glomeromycotan fungus occurred only in roots of H. rhombea. S. erythropa was the most dominant fungus found in the roots of H. rhombea. The detection of the same AM fungal species in field-collected roots of H. rhombea and R. parvifolius, which form Paris- and Arum-type AM, respectively, shows that AM morphology in these plants is strongly influenced by the host plant genotypes as appears to be the case in many plant species in natural ecosystems, although there are preferential associations between the hosts and colonizing AM fungi in this study.

Arum- and Paris-type arbuscular mycorrhizas in a mixed pine forest on sand dune soil in Niigata Prefecture, central Honshu, Japan.

Arbuscular mycorrhizas (AM) are the most widespread mycorrhiza in nature and form two morphologies, Arum- and Paris-type. The determining factors defining the two different morphologies are not well understood. In this study, the distribution of Arum- and Paris-type AM was determined in a mixed pine forest. A total of 35 plant species belonging to 20 families and 32 genera were identified and examined for AM colonization and morphological types. AM morphological types in 14 families were confirmed as follows: Arum-type in Rosaceae, Oleaceae, Lauraceae, Vitaceae and Compositae, Paris-type in Aquifoliaceae, Ulmaceae, Araliaceae, Theaceae, Magnoliaceae, Rubiaceae and Dioscoraceae, and both and/or intermediate types in Caprifoliaceae and Gramineae. Plant families whose AM morphological status was previously unknown were clarified as follows: Polygonaceae and Commelinaceae showed Arum-type morphology; Celastraceae, Menispermaceae and Elaeagnaceae had typical Paris-type morphology. The proportion of Arum-type to Paris-type species decreased in the following order: annuals > perennials > deciduous species > evergreen species, and pioneer group > early successional group > late successional group. Evergreen plants had a higher tendency to form Paris-type AM than annuals, perennials and deciduous plants. The results indicate that environmental changes, such as shade during plant succession, control the distribution of plant growth forms in mixed pine forest and may also play a part in the distribution of Arum- and Paris-type morphology. The identity of the plant seems to strongly influence AM morphology, though control by the fungal genome cannot be ruled out.