坂本 一憲

Soybean (Glycine max) roots establish associations with nodule-inducing rhizobia and arbuscular mycorrhizal (AM) fungi. Both rhizobia and AM fungi have been shown to affect the activity of and colonization by the other, and their interactions can be detected within host plants. Here, we report the transcription profiles of genes differentially expressed in soybean roots in the presence of rhizobial, AM, or rhizobial-AM dual symbiosis, compared with those in control (uninoculated) roots. Following inoculation, soybean plants were grown in a glasshouse for 6 weeks; thereafter their root transcriptomes were analyzed using an oligo DNA microarray. Among the four treatments, the root nodule number and host plant growth were highest in plants with dual symbiosis. We observed that the expression of 187, 441, and 548 host genes was up-regulated and 119, 1,439, and 1,298 host genes were down-regulated during rhizobial, AM, and dual symbiosis, respectively. The expression of 34 host genes was up-regulated in each of the three symbioses. These 34 genes encoded several membrane transporters, type 1 metallothionein, and transcription factors in the MYB and bHLH families. We identified 56 host genes that were specifically up-regulated during dual symbiosis. These genes encoded several nodulin proteins, phenylpropanoid metabolism-related proteins, and carbonic anhydrase. The nodulin genes up-regulated by the AM fungal colonization probably led to the observed increases in root nodule number and host plant growth. Some other nodulin genes were down-regulated specifically during AM symbiosis. Based on the results above, we suggest that the contribution of AM fungal colonization is crucial to biological N2-fixation and host growth in soybean with rhizobial-AM dual symbiosis.

The synergic effect of rhizobia (R) and arbuscular mycorrhizal (AM) fungi on the bioproduction, trace element partitioning and metallothionein gene expression of soybean-host was investigated under normal and elevated soil zinc conditions. In a completely randomized 3×4 factorial design, the experimental treatments – Zn addition (0, 200, and 400 mg Zn kg−1 soil) and inoculation (uninoculated control, R, AM, and RAM dual inoculation) were set up in the greenhouse for nine weeks. While the inoculants effectiveness was decreased in 400 mg Zn kg−1 soils, RAM induced significantly higher biomass production under all soil Zn treatments. The biomass response indicated that AM modulated stem and root bioproduction in favor of leaf/pod, while rhizobium favored root production and potentiated AM effect in dual inoculation. The partitioning of Zn and Mn in the hosts indicated synergic effects between AM and R, in RAM plants. Compared with control, AM lowered leaf Zn concentrations by reducing root Zn concentrations and modulating root-to-stem and stem-to-leaf Zn translocations. Compared to AM, RAM plants achieved lower leaf and pod Zn concentrations by mainly reducing root-to-stem Zn translocation. Zn treatment increased leaf and pod Mn in control plants, but symbionts countered this by regulating root-to-stem Mn translocation, especially in RAM. Type 1 metallothionein gene expression in roots was highest in RAM and lowest in control plants, but Zn effects were not dose-dependent. Synergisms in symbionts root colonization, number and greenness of leaves, element partitioning and metallothionein gene expression are indicated as important mechanisms underlying the effective partnership between AM and R, in the dual inoculation.

The effect of excess Zn and arbuscular mycorrhizal (AM) fungus on bioproduction and trace element nutrition were investigated in tomato. In a completely randomized factorial design, the experimental treatments–Zn addition at 0 (normal) and 300 (excess) mg Zn kg−1 soil, and AM inoculation (non-AM and Rhizophagus irregularis)–were set up in a growth chamber for 10 weeks. Generally, AM effects on the available Zn, Mn, Cu, and Fe in the rhizosphere soil were in tandem with the effects in host tissues. Under normal Zn condition, AM enhanced Cu availability in the rhizosphere, optimized the Cu:Zn balance in shoots, and increased the host biomass production. Excess Zn reduced mycorrhizal colonization in AM plants and the total plant biomass in both AM and non-AM plants. Although AM decreased the Zn concentrations in soil and host tissues under excess Zn, the distortions in host TE balance were not significantly ameliorated by the fungus. While Zn in fruit was within the safety threshold, Mn deficiency in the fruit was observed under excess Zn, alongside increased root-to-fruit Fe and Cu translocations. Mycorrhizal reductions in soil and tissue Mn concentrations were considered a minus in terms of probable symbiont amelioration of Mn:Zn in-balance under excess Zn. Additional microbe(s) that can enhance Mn homeostasis might be helpful in tomato under elevated soil Zn.

Root colonization by arbuscular mycorrhizal (AM) fungi has traditionally been analyzed by microscopy. However, this method is time consuming and it is often difficult to distinguish between AM and non-AM fungi. In this study, we analyzed the fatty acid profiles in soybean roots colonized by AM fungi to determine if specific fatty acids derived from AM fungi can be used as markers for the intensity of the AM fungal colonization. The wild-type Enrei and hypernodulating Kanto100 soybean cultivars were inoculated with an AM fungus (Gigaspora rosea) alone or with Bradyrhizobium diazoefficiens, which nodulates soybean roots. Fatty acids 20:1ω9, 20:4ω6, and 20:5ω3 were specifically detected in the lateral roots of AM fungus-inoculated and dual-inoculated soybean plants. In the second lateral roots, the percentage of AM-specific fatty acids (i.e., 20:1ω9, 20:4ω6, and 20:5ω3) derived from AM fungi was closely correlated with the intensity of the AM fungal colonization. We propose that the AM-specific fatty acids represent useful markers for estimating the degree of AM fungal colonization. The percentage of AM-specific fatty acids was more than twofold higher in the second lateral roots than in the first lateral roots. Thus, the degree of AM fungal colonization is probably twofold higher in the second lateral roots than in the first lateral roots.

The effect of Bradyrhizobia (R) and Arbuscular mycorrhiza (AM) on manganese, iron, phosphorus, and polyphenols was investigated in soybean cultivated in Zn treated soils. In a completely randomized 3 x 4 factorial, the experimental treatments - Zn addition (0, 200, and 400 mg Zn kg(-1) of soil), and Inoculation (uninoculated control, R, AM, and R+AM dual inoculation) were set up in the greenhouse. Zinc treatment increased Zn in roots and shoots, Fe in roots, frequency of root mycorrhization, P in root, and. Mn translocation; but decreased Fe translocation, Mn in root, and total polyphenols. Symbionts effected micronutrient balancing in the plants. Mycorrhizal inoculants increased Zn in shoots, and modulated Mn and Fe in shoots and roots; within the whole-plant crosstalk in Zn, Mn, and Fe status. In dual inoculation, synergy for much enhanced Mn translocation was indicated in 400 mg Zn kg(-1) treatment. Modulations of Zn-Mn-Fe nutrition in dual inoculation was supported with a reduction in cumulative number of fallen leaves, and increases in leaf greenness, shoot P, and root polyphenols, to give higher shoot biomass. Our results suggest the possibility of optimizing the crop production and bio-inoculation outcomes by targeting Mn and Fe status (in roots, shoots, translocation, and ratios with Zn) during cultivation under excess Zn. (C) 2017 Elsevier B.V. All rights reserved.

Phospholipid fatty acid (PLFA) and ester-linked fatty acid methyl ester (EL-FAME) extraction methods have been commonly used for estimating microbial biomass and characterizing microbial community composition in soil. However, to our knowledge, there is currently no comparison of these two methods across different ecosystems. A regression analysis for 8 different soils showed that there were significant linear relationships between PLFA and EL-FAME extracts (r(2) = 0.97, 0.98 and 0.72, respectively) for determining the fungal (18:20)6,9) and bacterial abundance and fungal-to-bacterial ratio (F:B ratio). However, regression lines of forest soils for fungal abundance and F:B ratio had a slight but significantly higher intercept than those of arable soils, i.e. higher EL-FAME:PLFA ratio for fungi in forests than those in arable soils. This might be due to that EL-FAME extracts may have additional factors such as physiological status of fungi and the inclusion of substantial amount of humic substances that affect quantitative determination of fungal biomass. Overall, EL-FAME method is simple and would produce similar results to PLFA method for bacteria in both quantitative and qualitative assessments when comparing different soils across ecosystems. However, for fungi, PLFA method would be more suitable than EL-FAME method.

Chitosan, a polymer of glucosamine, is one of the natural polysaccharides which are used as soil-amendment materials. We report here chitosan degradation and the corresponding effects on microbes in two distinctive types of soil. Having established a method to measure amounts of chitosan which was added to soil, the degradation of powdered chitosan in a sandy or a silty soil was investigated. In the chitosan-added sandy soil, chitosan amount and total carbon (C) content did not significantly change even after 30d. In contrast, chitosan added to the silty soil decreased to less than half of the initial amount (50mgg(-1) soil) after 10d incubation at 25 degrees C and seemed to be completely consumed after 30d. Total carbon and nitrogen (N) contents also declined according to the chitosan decrement, whereas the C/N ratio did not alter. Ammonium nitrogen was increasing over 0.7mgNg(-1) soil, during the 11d after the addition of chitosan. 16S rRNA gene-based polymerase chain reaction (PCR)-denaturant gradient gel electrophoresis (DGGE) analysis revealed that addition of chitosan caused drastic alteration of bacterial community structure in the silty soil, in contrast to fewer changes in the sandy soil. Nucleotide sequence analysis following DGGE revealed that actinobacteria belonging to genera Streptomyces and Kitasatospora strongly increased within 4d after the addition of chitosan to the silty soil. Seven out of eight chitosan-degrading bacteria, isolated from the silty soil 10d after the addition of chitosan, were identified to be Streptomycetes, based on the nucleotide sequence of the 16S rRNA gene. Among them, two strains, AMI7 and AMI10, exhibited identical nucleotide sequences with those detected in the PCR-DGGE analysis. The data strongly implied that those atcinobacterial genera, Streptomyces and probably Kitasatospora, are involved in the initial degradation process of chitosan in the silty soil. The presence and quantity of these genera may influence the chitosan degradation rate in soil.

マメ科植物には根粒菌とアーバスキュラー菌根菌（以下菌根菌）が二重に共生している。著者はふたつの共生系を同時に維持している宿主植物の代謝機構に興味を持ち，これまでに明らかにされたマメ科植物の根粒・菌根共生系に関与する宿主遺伝子の共通性と特異性に関する知見を整理した。90年代初めにマメ科モデル植物（ミヤコグサ・タルウマゴヤシ）の利用が提唱され，分子遺伝学的研究のための基盤整備が進められた。その結果，根粒菌または菌根菌が生産するシグナル物質を処理する宿主植物の初期シグナル伝達系に共通過程が存在することが明らかにされた（共通シグナル伝達系）。マメ科植物は過剰な根粒着生を抑制するオートレギュレーション機構を有していることが知られている。2000年にオートレギュレーションが破綻した根粒超着生変異体における菌根菌の樹枝状体形成が過剰であることが報告され，これが発端となってマメ科植物のオートレギュレーション機構は根粒着生のみならず菌根形成もコントロールしていることがあきらかとなった。マメ科植物の根粒共生系と菌根共生系における宿主遺伝子のトランスクリプトーム解析が行われている。著者はダイズの根粒・菌根共生系について検討し，ダイズは根粒菌と菌根菌のそれぞれに特異的な遺伝子を発現し，両共生菌を同時にかつ巧妙に制御していることを明らかにした。両共生系に共通して発現する遺伝子はその数は少なかったものの，ダイズが微生物と共生関係を結ぶ際の必須遺伝子であると考えられ，また進化的に菌根共生系から根粒共生系ヘ引き継がれた遺伝子であると推測された。

Flooding is one of the serious problems for soybean plants because it inhibits growth. Proteomic and metabolomic techniques were used to determine whether proteins and metabolites are altered in the root tips of soybeans under flooding stress. Two-day-old soybean plants were flooded for 2 days, and proteins and metabolites were extracted from root tips. Flooding-responsive proteins were identified using two-dimensional- or SDS-polyacrylamide gel electrophoresis-based proteomics techniques. Using both techniques, 172 proteins increased and 105 proteins decreased in abundance in the root tips of flood-stressed soybean. The abundance of methionine synthase, heat shock cognate protein, urease, and phosphoenol pyruvate carboxylase was significantly increased by flooding stress. Furthermore, 73 flooding-responsive metabolites were identified using capillary electrophoresis-mass spectrometry. The levels of gamma-aminobutyric acid, glycine, NADH2, and phosphoenol pyruvate were increased by flooding stress. Taken together, these results suggest that synthesis of phosphoenol pyruvate by way of oxaloacetate produced in the tricarboxylic acid cycle is activated in soybean root tips in response to flooding stress, and that flooding stress also leads to modulation of the urea cycle in the root tips.

Soybean plants autoregulate to suppress excessive nodulation. It has been revealed recently that the autoregulation of various legumes controls both nodulation and arbuscular mycorrhizal (AM) fungal colonization. We investigated the involvement of autoregulation in the interaction between rhizobial nodulation and AM fungal colonization. We used a wild-type soybean cv. Enrei and its hypernodulating mutant Kanto100, defective in the autoregulation. We included four different treatments: an uninoculated control, inoculation with rhizobium Bradyrhizobium japonicum alone, inoculation with AM fungus Gigaspora rosea alone, and dual inoculation with rhizobium and AM fungus. In both Enrei and Kanto100, AM fungal colonization enhanced the weight and N-2 fixation of nodules, suggesting that autoregulation of host plant is not involved in the stimulatory effect of AM fungal colonization on rhizobial nodulation. In plants with the AM fungus alone, the AM fungal colonization of Enrei was comparable to that of Kanto100. In plants with dual inoculation, however, this was significantly (P < 0.05) lower than in Kanto100. To confirm the control of AM fungal colonization by the autoregulation of host plant, a reciprocal grafting experiment was performed between Enrei and Kanto100. In plants with the AM fungus alone, AM fungal colonization was comparable among Enrei (shoot)/Enrei (root), Enrei/Kanto100, Kanto100/Enrei, and Kanto100/Kanto100 grafts. In plants with dual inoculation, however, AM fungal colonization of Enrei/Enrei and Enrei/Kanto100 grafts was significantly (P < 0.05) lower than that of Kanto100/Enrei and Kanto100/Kanto100. These results indicate that rhizobial nodulation suppresses AM fungal colonization, and the autoregulation of host plant, initiated by nodulation, is involved in this phenomenon.

Laccase oxidizes iodide to molecular iodine or hypoiodous acid, both of which are easily incorporated into natural soil organic matter. In this study, iodide sorption and laccase activity in 2 types of Japanese soil were determined under various experimental conditions to evaluate possible involvement of this enzyme in the sorption of iodide. Batch sorption experiment using radioactive iodide tracer (I-125(-)) revealed that the sorption was significantly inhibited by autoclaving (121 degrees C, 40 min), heat treatment (80 and 100 degrees C, 10 min), gamma-irradiation (30 kGy), N-2 gas flushing, and addition of reducing agents and general laccase inhibitors (KCN and NaN3). Interestingly, very similar tendency of inhibition was observed in soil laccase activity, which was determined using 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) as a substrate. The partition coefficient (K-d: mL g(-1)) for iodide and specific activity of laccase in soils (Unit g(-1)) showed significant positive correlation in both soil samples. Addition of a bacterial laccase with an iodide-oxidizing activity to the soils strongly enhanced the sorption of iodide. Furthermore, the enzyme addition partially restored iodide sorption capacity of the autoclaved soil samples. These results suggest that microbial laccase is involved in iodide sorption on soils through the oxidation of iodide.

Bradyrhizobium sp. S23321 is an oligotrophic bacterium isolated from paddy field soil. Although S23321 is phylogenetically close to Bradyrhizobium japonicum USDA110, a legume symbiont, it is unable to induce root nodules in siratro, a legume often used for testing Nod factor-dependent nodulation. The genome of S23321 is a single circular chromosome, 7,231,841 bp in length, with an average GC content of 64.3%. The genome contains 6,898 potential protein-encoding genes, one set of rRNA genes, and 45 tRNA genes. Comparison of the genome structure between S23321 and USDA110 showed strong colinearity; however, the symbiosis islands present in USDA110 were absent in S23321, whose genome lacked a chaperonin gene cluster (groELS3) for symbiosis regulation found in USDA110. A comparison of sequences around the tRNA-Val gene strongly suggested that S23321 contains an ancestral-type genome that precedes the acquisition of a symbiosis island by horizontal gene transfer. Although S23321 contains a nif (nitrogen fixation) gene cluster, the organization, homology, and phylogeny of the genes in this cluster were more similar to those of photosynthetic bradyrhizobia ORS278 and BTAi1 than to those on the symbiosis island of USDA110. In addition, we found genes encoding a complete photosynthetic system, many ABC transporters for amino acids and oligopeptides, two types (polar and lateral) of flagella, multiple respiratory chains, and a system for lignin monomer catabolism in the S23321 genome. These features suggest that S23321 is able to adapt to a wide range of environments, probably including low-nutrient conditions, with multiple survival strategies in soil and rhizosphere.

コウライシバを植栽した屋上緑化薄層基盤軽量土壌の経年変化を調査した。芝の生育，土壌物理性に経年的な変化は見られなかった。土壌有機物は施工後から増加する傾向にあり，栄養塩類含有量は，ゴルフ場グリーンの土壌診断基準値とほぼ同等であった。また，未使用の軽量土壌中の栄養塩類量は施工後の含有量より有意に高く，施工後は経年的に減少した。追肥の有無による栄養塩類量の差は確認されなかった。これらのことから，屋上独自の環境を考慮し，その管理目標に適した施肥管理を行う必要があることが明らかになった。

Soil-inhabiting fungal pathogen Fusarium oxysporum often causes severe yield losses in many crops. We investigated the effect of a plant growth-promoting fungus, Penicillium sp. EU0013 on Fusarium wilt disease. In dual culture experiments, EU0013 inhibited the growth of Fusarium wilt pathogens by producing an inhibition zone. In experiments using sterile potting medium under controlled conditions, EU0013 significantly reduced the severity of Fusarium wilt on tomato (Solanum lycopersicum L.) and cabbage (Brassica oleracea L. var. capitata). In non-sterile soil, benomyl-resistant mutants of EU0013 were selected by exposing the conidial solution of EU0013 to ultraviolet light. The selected mutant EU0013_90S isolate did not show any distinct differences from EU0013 in colony characteristics, growth rate or antifungal activity against Fusarium wilt pathogens in dual culture. The effect of EU0013_90S on tomato wilt was studied under greenhouse conditions using non-sterile soil. Two-weeks old tomato seedlings were dipped in four different concentrations of EU0013_90S conidial suspension (1 x 103, 1 x 104, 1 x 105, and 1 x 106 conidia mL-1). Seedlings were then planted in soil inoculated with either F. oxysporum f. sp. lycopersici race 1 CU1 or race 2 JCM 12575 (1 x 106 bud-cells g-1). We found the greatest disease suppression occurred when seedlings were dipped in the highest concentration of EU0013_90S conidia. This same inoculum concentration of EU0013_90S also resulted in the highest disease reduction in soil infested with JCM 12575. Higher root colonization with EU0013_90S showed a significant reduction in Fusarium wilt disease, suggesting that colonization by Penicillium sp. EU0013_90S is important for efficient biocontrol of these diseases.

Modification of forest vegetation caused by an overabundance of mammalian herbivores has been reported in temperate and subarctic regions all over the world. However, the indirect effects of these herbivores on the structure and functions of soil decomposer systems are not fully understood, especially in temperate forests. We investigated the early effects of sika deer invasion on soil decomposer systems in a Japanese temperate forest using two large-scale experimental enclosures with low and high densities of deer (LD: 25 ha, 4 deer km(-2): HD: 6.25 ha, 16 deer km(-2)) including control plots without deer (WD). Three years after deer introduction the understory cover of dwarf bamboo (Sasa nipponica) declined due to deer browsing in both enclosures. At the same time, measurements were made of the soil microbial community, soil nematode community, soil nitrogen (N) mineralization rate, and carbon (C) and N content in dwarf bamboo leaves. In LD, soil microbial biomass was lower from WD, probably due to the decrease of fresh aboveground litter from dwarf bamboo. Surprisingly, there were no cascading effects on total abundance of soil nematodes and soil total N mineralization potential which were unaffected by deer in the LD treatment, while soil NH(4)(+)-N content was lower and soil nematode community structure was different (abundance of 4 families was higher and that of 3 families was lower, but the functional structure was not different) from WD. Specifically, the responses to deer introduction varied between microbes and nematodes, and the change of balance in the microbial food webs may have altered N mineralization processes. In contrast, in the HD treatment, all the variables measured were not significantly different from those of WD treatment. Intensive browsing by deer may have cancelled out the effects of the decrease in aboveground litter input on the soil decomposer systems through other pathways, such as a transitory increase in belowground litter input caused by induced changes in allocation patterns of bamboo. No changes in total N mineralization potential, leaf N, and composition of understory vegetation in both enclosures indicated that deer introduction did not facilitate nor retard N cycling regardless of deer density. This study showed that sika deer browsing can affect soil decomposer systems at an early stage of invasion even at low density, which contrasts with previous studies on the subject. Linking our findings of early-stage effects of deer on soil decomposer systems to longer-term dynamics of understory vegetation and tree regeneration will be needed to evaluate the adequacy of deer management practices with respect to the sustainability of soil nutrient supplies. (C) 2010 Elsevier B.V. All rights reserved.

Biocontrol as an alternative strategy for management of tomato wilt using a plant growth promoting fungus(PGPF), Penicillium sp. EU0013, isolated from eucalyptus roots was investigated under growth chamber conditions.Roots of tomato seedlings were dipped in different inoculum concentrations of EU0013 followed by Fusariumoxysporum f. sp. lycopersici race 1 CU1( Fol)( 0, 104, 105, 106 and 107 conidia ml-1), respectively. Treatmentof tomato seedlings roots with EU0013 resulted in significant disease reduction( 49% to 84%) depending on differentEU0013 and Fol conidial concentrations used. The highest disease reduction (84%) occurred in the treatment,where EU0013 and Fol were applied at lowest inoculum's concentration of 104 conidia ml-1, compared withits high conidial concentration. The plant biomass data indicated that fresh shoot and root weight of tomato seedlingstreated prior with EU0013 were significantly higher than the tomato plants treated with Fol only. The highestshoot weight (1.10 g) and root weight (0.75 g) were recorded in the treatment, where roots were treated onlywith EU0013( 107 conidia ml-1). Our results suggest that PGPF, Penicillium sp. EU0013 is capable of enhancinggrowth and protecting tomato plants against Fusarium wilt under growth chamber conditions.

Volcanic acidification has created unique ecosystems that have had to adapt to the acidic environments in volcanic regions. To characterize the primary microbial properties of strongly acidified soils in such environments, we investigated microbial biomass, nitrogen transformations and other relevant chemical properties in the surface soils of solfatara and forests from Osorezan, a typical volcanic region in Japan, and compared the results to common Japanese forest soils. Soil microbial biomass C (MBC) and N (MBN) were determined using the chloroform fumigation-extraction method. Potential net N mineralization and net nitrification were measured in aerobic laboratory incubations. Long-term acidification in the Osorezan soils by volcanic hydrogen sulfide deposition caused low soil pH (3.0-3.8), base cation deficiency and increased concentrations of toxic ions such as Al(3+). The proportions of MBC to total carbon (MBC/TC ratio) and MBN to total nitrogen (MBN/TN ratio) were lower than those in common Japanese forest soils. The extreme acidic conditions may have inhibited microbial survival in the Osorezan acid soils. Net N mineralization occurred at rates comparable to those in common Cryptomeria japonica forest soils, probably because of the presence of acid-tolerant soil microorganisms. Net nitrification was completely inhibited and autotrophic ammonia oxidizers were not detected by the MPN method. The inhibition of nitrification prevents nitrogen leaching from the soils, thus maintaining a nitrogen cycle in the volcanic acid region in which NH(4)(+) (and NH(3)) is recycled among microorganisms and plants.

Fusarium wilt is caused by soil-borne pathogen Fusarium oxysporum. Tomato (Lycopersicon esculentum Mill.) is susceptible to Fusarium oxysporum f. sp. lycopersici race 1 and was infected with wilt disease. A pot experiment was conducted to investigate effects of inoculating arbuscular mycorrhizal (AM) fungus (Glomus etunicatium) on the microbial community in the rhizosphere soil and Fusarium wilt in tomato (cv. Oogatafukuju). The results indicated that AM fungal inoculation suppressed the Fusarium number in the rhizosphere soil of tomato and decreased the Fusarium wilt disease index. Compared to the control, AM fungal inoculation increased the actinomycete number but increased bacterial number. Bacterial and fungal numbers were high but actinomycetes number was low when tomato basal stems became discolored brown. Fusarium inoculation significantly suppressed development of AM colonization and decreased polyphenol oxidase (PPO) activity in leaves and roots of tomato. Inoculation with AM fungi and Fusarium maintained high PPO activity in leaves and roots. The AM colonization increased root growth of tomato, whereas Fusarium inoculation had no significant effect on tomato growth. These findings suggest that because AM fungal inoculation changes microbial communities and enhances PPO activity, it should suppress occurrence of Fusarium wilt in tomato.

The roots of soybean (Glycine max [L.] Merr.) establish symbiosis with nodule-inducing rhizobia and arbuscular mycorrhizal (AM) fungi. The existing nodules systemically suppress subsequent nodule formation, a phenomenon known as autoregulation. Grafting experiments revealed that some forms of autoregulation are controlled by the shoot. In the present study, we examined shoot-controlled regulation of AM fungal colonization using a reciprocal grafting technique. Ten-day-old seedlings of wild-type soybean cv. Enrei and its hypernodulating mutant En6500 were cut below the cotyledons and the shoots were grafted to self or reciprocal roots. Grafted seedlings were inoculated with Bradyrhizobium japonicum and Gigaspora rosea and grown in a glasshouse for 60 days. The arbuscule abundance of the En6500 (shoot)/En6500(root) graft was 1.5-fold higher than that of the Enrei/Enrei graft. In grafts between Enrei and En6500, an increased arbuscule abundance was detected only when En6500 was used as the shoot. The arbuscule abundance of Enrei/En6500 when Enrei was used as the shoot was comparable to that of Enrei/Enrei. The intensity of AM fungal colonization was lower in Enrei/En6500 than in the other grafting treatments. From the results obtained, we suggest that soybean shoots systemically control arbuscule formation in both AM symbiosis and nodule formation.

千葉県房総半島の中西部はわが国最大の山砂採取地である。県内の山砂生産量は1968年から累計すると約6億8,000万m3に上り、全国の山砂生産量のうち約51％が千葉県から産出されている。2007年に山砂採取を行っている事業所は174カ所、その合計面積は約1,410haに達している。山砂を埋蔵する地層は上総層群と呼ばれ、東京湾から内陸部へおよそ幅3km、長さ36kmにわたって帯状に分布しており、これに沿って山砂採取地が点在している。産出された山砂は埋立てやコンクリートの骨材に使われ、東京湾岸等の開発に大きく貢献してきたが、その一方で植生が乏しい広大な荒廃裸地を生み出している。山砂採取事業は砂利採取法と森林法によって開発後の緑化が義務付けられており、早期の植生回復が必要とされている。しかし跡地の表土は主に山砂残土と下層土からなり、非常に貧栄養であるため植林しても樹木の生育は非常に遅い。また跡地は風雨による土壌侵食が激しく、樹木の生育を妨げ2次災害や景観の悪化などの問題を招いている。本研究は、グランドカバー植物として既に法面や畦畔の緑化に用いられているセイヨウミヤコグサを山砂採取跡地において生育させるために、牛ふん堆肥の適切な施用量について検討した。これは貧栄養である跡地土壌で植物を生育させるためには、肥料や土壌改良資材となる有機物の施用が不可欠であるためである。またAM菌の接種実験も試み、セイヨウミヤコグサの生育に及ぼすAM菌の影響と植物根の菌根形成率に及ぼす牛ふん堆肥の影響について考察した。

Soil microbial communities in an apple orchard and its adjacent boundary bush with or without infestation by violet root rot were investigated for 2 years. Effects of season (spring, summer, and fall), land-use (apple orchard and boundary bush), and violet root rot (infested and healthy) on soil microbial populations, microbial activity, and microbial community structures were determined using physiological, cytochemical, and molecular (PCR-DGGE) approaches. Seasonal fluctuations were significant (P<0.05) in viable bacteria and fungal populations, bacterial FAME, fluorescein diacetate (FDA) hydrolysis, and diversity (H') and evenness (Y) of community-level physiological profile (CLPP) in both years. However, seasonal differences of soil microbial guilds that utilize carbon substrate groups observed in the first year were not reproduced in the second year. The land-use factor differentiated the apple orchard from the boundary bush where viable bacterial population, bacterial FAME and FDA hydrolysis were significantly greater in both years. Infestation status of violet root rot, on the other hand, significantly increased bacterial FAME and FDA hydrolysis in both years. In addition, neither the land-use nor the disease infestation factor significantly influenced the utilization patterns of individual substrate guilds for the 2 years. In both years, saturated fatty acids were significantly more abundant in the orchard than in the bush soil, and monosaturated fatty acids vice versa. Principal component analyses for CLPP, FAME, and denaturing gradient gel electrophoresis (DGGE) consistently exhibited that, although the violet root rot influenced the soil microbial community structures both in the apple orchard and the boundary bush, overall magnitude of the difference in communities between the violet root rot infested and non-infested sites in the bush were greater than in the orchard, irrespective of the season. These results suggested that the seasonal and the land-use factors affected soil microbial community both quantitatively and qualitatively, whereas the impact of the violet root rot on the soil microbial community was mainly qualitative and more pronounced in the adjacent bush than in the orchard. (C) 2008 Elsevier Ltd. All rights reserved.

The effects of low densities of native browsing mammals on nutrient cycling are not fully understood. Weak browsing may improve nitrogen (N) mineralization in soil and positively affect plant regrowth at the forest floor. To investigate the effects of weak browsing by sika deer (Cervus nippon) oil soil subsystems, we defoliated a dwarf bamboo (Sasa nipponica)-dominated understory layer in a natural forest at different intensities to realistically simulate deer browsing. Defoliation (0-18% leaf removal) was performed three times at approximately 1-week intervals in summer. We measured water-soluble carbon (C) concentration, phospholipid fatty acid (PLFA) profiles as indicators of microbial community structure, a PLFA of 20:4 as an indicator of protozoan abundance, nematode community structure at the family level, and the N mineralization rate in 28 days of incubation. The effects of defoliation oil each soil parameter were determined by comparing before and after defoliation values. The N mineralization rate in the first 10 days of incubation showed a unimodal response to defoliation intensity, with a peak mineralization rate at a defoliation rate (number of removed leaves/total leaves) of 7.6%, correlating with protozoan PLFA and the abundance of Plectidae (the most dominant family of bacterivorous nematodes). In contrast, the N mineralization rate during the following 18 days of incubation decreased monotonically with increasing defoliation intensity, correlating with the water-soluble C concentration in the soil and the C content of new leaves. These results suggest that removing 15% of leaves may have induced a pulse-like release of labile organic matter from roots that lasted for less than I week and stimulated N mineralization through microbial loop in soil in the short term (in the first 1-2 weeks after defoliation). N mineralization, however, was reduced with increase of defoliation intensity in the longer term (3-5 weeks after defoliation), possibly because of the reduction in labile organic matter supply from roots I week after defoliation. As a result, N mineralization rates over the 28-day incubation period responded to defoliation intensity in a unimodal pattern with a small peak (at a defoliation rate of 4.9%) and were negatively affected by high defoliation rates (> 10%). This study suggests that browsing on forest floor plants has positive or negative effects on soil N mineralization potential depending on browsing intensity level. (c) 2007 Elsevier Ltd. All rights reserved.

The potentials of nitrous oxide (N(2)O) and carbon dioxide (CO(2)) production in acid tea soils from Indonesia and Japan were investigated in a laboratory incubation experiment, and the community structures of ammonia-oxidizing bacteria in these soils were characterized using PCR-DGGE approaches. The soils used were sampled from tea plantations in Shizuoka, Japan and in Bogor and Malino, Indonesia. All of the soils were acidic (pH 3.45 to 4.00). The N(2)O and CO(2) production in Shizuoka was almost 5 times higher than in Bogor and Malino. All of the amoA gene sequences defined belong to the genus Nitrosospira sp. with cluster 2 and cluster 3a.

AM菌の接種および木炭の施用が豚ぷんコンポストに由来するZnとCuの作物吸収と地上部への移行に及ぼす影響を検証するため,トウモロコシを用いてポット試験を行った.またMIDIシステムを用いて土壌脂肪酸を分析し,AM菌のプロパギュール測定における有効性を考察した.前年に豚ぷんコンポストを施用した土壌に木炭施用の有無およびAM菌(Glomus etunicatumおよびGlomus sp.CB0301)接種の有無により6栽培試験区を設けた.その結果,以下のことが明らかとなった.1)各試験区のZnとCuの土壌含有量は試験開始時と試験終了時との間に顕著な差はなく,作物吸収,AM菌接種および木炭施用による減少は確認できなかった.2)作物のZn含有率は木炭施用の有無にかかわらずAM菌を接種した作物体地上部で顕著に増大(26〜42%)した.一方,Cuの地上部への移行は少なかった.3)MIDIシステムを用いた土壌脂肪酸分析においてAM菌特異的脂肪酸である16:1ω5をはじめ各種脂肪酸の検出および定量が可能であった.4)土壌中の脂肪酸はAM菌を接種した試験区でより多くの種類が検出され,AM菌に特異的な16:1ω5の含有割合が最も高かった.また,土壌中の脂肪酸含有量はGlomus sp.CB0301接種区で顕著に増大し,AM菌の接種と木炭の施用を同時に行った試験区で更に増大した.5)木炭施用がトウモロコシの菌根形成に及ぼす影響は不明確であったが,土壌では16:1ω5量およびAM菌胞子数の間に有意な正の相関関係があった.以上の結果より,AM菌は宿主植物への豚ぷんコンポスト由来のZn供給を促進すること,また木炭施用は宿主における菌根形成の促進よりも,土壌中におけるAM菌プロパギュールの増殖(外生菌糸や胞子のバイオマスの増加)に寄与することが考えられた.また,MIDIシステムを用いた土壌脂肪酸の分析がAM菌のプロパギュールの測定に有効であることが明らかとなった.

牛ふんコンポスト,豚ぷんコンポストおよび化学肥料を各種畑土壌(褐色低地土,多腐植質黒ボク土,褐色森林土)にそれぞれ5年間連用した場合に栽培されたコマツナについて地上部の亜鉛,クロム,マンガンの含有率について調べた.亜鉛,クロム,マンガンの測定には,高感度な全量同時分析を行うために機器中性子放射化分析法(INAA)を用いた.その結果,以下の知見が得られた.1)INAAによるコマツナ地上部の亜鉛とクロムの含有率を硝酸分解法による測定値と比較した結果,亜鉛では硝酸分解法による測定値はINAAによる測定値とほぼ同じであったが,硝酸分解法によるコマツナ地上部のクロム含有率はINAAの場合の13〜27%であった.このため,INAAによる全量分析は作物による吸収量を正確に把握するために有用であると考えられた.2)牛ふんコンポスト施用によって,褐色低地土で栽培したコマツナの地上部のクロムの含有率,褐色森林土で栽培したコマツナの地上部の亜鉛含有率が増加した.しかし,多腐植質黒ボク土で栽培したコマツナの地上部のクロムとマンガンの含有率や褐色森林土で栽培したコマツナの地上部のクロムとマンガンの含有率は牛ふんコンポストの施用によって低下した.一方,豚ぷんコンポスト施用によって,褐色低地土で栽培したコマツナの地上部の亜鉛含有率,黒ボク土で栽培したコマツナの地上部の亜鉛とマンガンの含有率,褐色森林土で栽培したコマツナの地上部の亜鉛,クロム,マンガンの含有率が増加した.しかし,褐色低地土で栽培したコマツナの地上部のクロム含有率は豚ぷんコンポストの施用によって低下する傾向を示した.褐色低地土の牛ふんコンポスト施用区と豚ぷんコンポスト施用区のクロム,多腐植質黒ボク土の牛ふんコンポスト施用区のクロムとマンガン,褐色森林土の牛ふんコンポスト施用区の亜鉛,クロム,マンガンについては,家畜ふんコンポスト中の亜鉛,クロム,マンガンのコマツナによる利用性の変化がコマツナによるこれらの元素の吸収に影響を及ぼしている可能性が考えられた.3)3土壌の家畜ふんコンポスト施用区における亜鉛,クロム,マンガンのコマツナ地上部の吸収率は亜鉛で1〜6%,クロムで2〜55%,マンガンで2〜12%であった.家畜ふんコンポスト施用区では5年間の施用歴があるにもかかわらず土壌中のクロムやマンガンの含有量は亜鉛と異なり化学肥料施用区を上回らなかった.従って,クロムやマンガンの土壌蓄積性を明らかにするためには今後更にデータを蓄積していくことが重要であり,土壌調査を継続して行う必要がある.

The response of soil carbon availability and microbial utilization efficiency to varying vegetations and to increasing temperature emphasizes the need for further research in volcanic forest soils. We have studied responses of soil microbial biomass C (MBC) concentrations and metabolic quotients (qCO(2) CO2-C/biomass-C) to vegetations and temperature in temperate volcanic forest soils. Soils were sampled in April and October 2003 beneath four forest stands in close proximity (200 m apart) in central Japan: a Japanese cedar coniferous forest (CI), a pine coniferous forest (CII), and an oak-dominated hardwood on a grading and steep slope (DI and DII). The soil MBC concentrations and qCO(2) values beneath each forest stand were measured under oxic conditions, along with the effect of temperature and the long-term storage at low temperature on both variables. Comparing different forest stands, it was indicated that the pine forest soil always showed the lowest MBC concentration relative to soil total C, and the largest qCO(2) (P < 0.05), suggesting that microbial communities in the soil were less efficient in C use than communities in the other forest soils. Under a same oak-dominated hardwood, a steep terrain induced a lower soil respiration and MBC concentration, but qCO(2) values of both soils had no significant differences at 25 C. The MBC concentration in the soils beneath each forest stand mostly had no differences during two sampling periods. The low-temperature storage induced an increase in soil MBC and a reduction of CO, production, reducing the qCO(2). There was a smaller MBC concentration in the CII soil than in the CI and DII soil within the range of 5 to 35 C; the MBC concentration and qCO(2) in the DI and CII soil appeared more sensitive at increasing temperature than those in the DII and CI soil. From the results obtained, it can be concluded that under similar climatic conditions and soil types, the MBC concentrations and qCO(2) values of temperate volcanic forest soils vary with tree species and temperature, and that the high soil C-to-N ratios and low acidity can contribute to decreased soil MBC concentration and to increased qCO(2). (c) 2006 Elsevier B.V. All rights reserved.

We measured the concentrations of atmospheric acidic pollutants at Mt. Tsukuba using a newly developed portable filter pack sampler. The results were analyzed to characterize the distribution and origin of pollutants, and were compared with measurements from Tsukuba City and from other mountainous areas in Japan.

An experiment was conducted to determine the accumulation of trace metals, zinc (Zn), chromium (Cr), manganese (Mn) and iron (Fe), in three types of arable soils (Brown Lowland soil, Andosol and Brown Forest soil) amended with chemical fertilizers (CHF), cattle farrnyard manure (CFM) and pig farmyard manure (PFM) over a 5-year period. Two methods were used to determine the total amount of trace metals: the instrumental neutron activation analysis (INAA), as a non-destructive measurement, and the concentrated acid digestion method (CADM), as a destructive measurement. Results of soil analyses using both INAA and CADM indicated that PFM application resulted in a considerable increase in the amount of Zn in arable soils compared with CHF application, unlike CFM application. Results obtained from the two methods were not fully in agreement for Cr. Based on INAA, soil accumulation of Cr from CFM and PFM applications was not significant in any soil type. In contrast, based on CADM, Cr accumulation was relatively higher in the Brown Forest soil with PFM than with CHF application. The soil types and treatments exerted various effects on Cr, Mn and Fe concentrations. The 5-year application of animal manure did not result in a considerable increase in Cr, Mn and Fe concentrations in soils, whereas longer-term application may lead to a gradual accumulation of Cr and Fe depending on the soil and manure types. The use of CADM in the present study may not always be adequate to assess the accumulation of trace metals in soils amended with CFM and PFM because of the conditions of the measurements, such as sensitivity, dilution level or type of instrument, and not because of the digestion method. The types of soil and manure affect trace metal accumulation in arable soils, and elements in the manure relate to the ability of soils to retain trace metals. It is important that the results of non-destructive and destructive measurements, obtained using INAA and CADM, be evaluated properly.

エンバク根およびユーカリ根から根部定着糸状菌を分離して,キャベツ苗に対して発芽促進および生育促進効果を持つ菌株を選抜することを試みた.その結果,糸状菌を接種しないキャベツ種子は播種後4〜6日に分散して発芽したのに対し,エンバク根から分離されたMT0011株とユーカリ根から分離されたEU0013株を接種した種子は播種4日目に集中して発芽した.また両糸状菌を接種した種子は最終発芽率に達する期間が短縮された.分離菌のキャベツ苗に対する生育促進効果について接種試験を3回繰り返して検討した.1回目の試験では供試した29菌株中7菌株がキャベツ苗の生育を促進した.2回目の試験では,MT0008株とEU0013株の2菌株が引き続き促進効果を示した.この2菌株について3回目の試験を行ったところ,EU0013株のみが促進効果を示した.接種試験に供試した菌株から坂本・萩原が分離した2菌株を除いた27菌株についてrDNA ITS領域の塩基配列による分子系統解析を行ったところ,1菌株を除いたすべてが子のう菌類に分類された.発芽促進効果を示したMT0011株はそのrDNA ITS領域の塩基配列の結果からDrechelaに近縁な菌株であると考えられた.また発芽促進と生育促進の両方の効果を示したEU0013株は分生子柄の形態と塩基配列の結果からPenicillium属であると同定された.

2種類の下水汚泥コンポスト (オガクズ汚泥コンポスト, モミガラ汚泥コンポスト) を3年間施用した褐色森林土の畑圃場における栽培作物 (ニンジンとエンバク) の各種元素吸収を化学肥料のみを施用した場合と比較した。その結果, 次のことが明らかになった。<BR>1) オガクズ汚泥コンポストの施用によってエンバクにおいては茎葉部のZnとBr, 根部のMn, Zn, Ag, Ba, 穂のClとZn, ニンジンにおいては茎葉部のNa, Sc, Mn, Fe, Co, Sm, 地下部の皮のMg, Sc, Mn, Zn, Br, Ba, La, 地下部の可食部のMn, Fe, Co, Znの含有率が増加した。<BR>2) モミガラ汚泥コンポストの施用によってエンバクにおいては茎葉部のK, Cr, Mn, Zn, Br, 根部のMn, Zn, Ag, 穂のZn, Br, ニンジンにおいては茎葉部のNa, Mn, Zn, Br, Sm, 地下部の皮のMg, Mn, Br, 地下部の可食部のCl, Mn, Zn, Brの含有率が増加した。

We developed a method to analyze atmospheric SO, (particulate SO42- + gaseous SO2) and NOx (NO + NO2) simultaneously using a battery-operated portable filter pack sampler. NOx determination using a filter pack method is new. SO2 and NOx were collected on a Na2CO3 filter and PTIO (2-phenyl-4,4,5,5-tetramethylimidazoline-3-oxide-1-oxyl) + TEA (triethanolamine) filters (6 piled sheets), respectively. Aqueous solutions were then used to extract pollutants trapped by the filters and the resulting extracts were pre-cleaned (e.g. elimination of PTIO) and analyzed for sulfate and nitrite by ion chromatography. Recoveries Of SO2 and NOx from standard pollutant gases and consistency of the field data with those from other instrumental methods were examined to evaluate our method. SO, and NO, could be analyzed accurately with determination limits of 0.2 ppbv and 1.0 ppbv (as daily average concentrations), respectively. The sampler can determine SOx and NOx concentrations at mountainous or remote sites without needing an electric power supply.

An experiment was conducted to examine the accumulation and mobility of heavy metals (Zn and Cu) at different depths in three types of arable soils (Brown Lowland soil, Andosol, and Brown Forest soil) amended with cattle and pig farmyard manures for 5 years. Nitric-perchloric acid digestion was performed for the determination of the total amounts of heavy metals, and 0.1 m hydrochloric acid extraction was performed for the determination of the amounts of soluble heavy metals. Results of the soil analysis indicated that pig farmyard manure application resulted in serious contamination of arable soils with Zn and potentially Cu. Especially, the Brown Forest soil displayed a high ability to accumulate heavy metals on the soil surface. Total-Zn concentration in surface soils was considerably affected by the holding capacity of soluble-Zn fraction. Although the Andosol amended with pig farmyard manure showed higher concentrations of heavy metals related to the higher ability of retention on a weight basis, the soil did not contribute to high heavy metal accumulation because of its low bulk density. Heavy metals were easily leached in sandy soils such as Brown Lowland soil, and Cu was potentially stable compared with Zn. We suggest that long-term pig farmyard manure application to the Brown Lowland soil and Andosol with a light soil texture is associated with a higher risk of groundwater pollution than the application to the Brown Forest soil.

The soil microbial biomass is important such as pool of plant nutrients and is also driving force of the cycling of C, N, P and S in soil. However, the microbial biomass in acid soil has not been fully investigated due to the limitation of methods, i.e. chloroform-fumigation incubation or substrate-induced respiration because of decreased basal mineralization in chloroform-fumigated soil under acid conditions. This paper reviews improvement and application of these methods and vertical distribution of microbial biomass in two kinds of acid soils; namely, Andisols as dominant upland soils in Japan and tropical peat soils as potentially important lowland soils for agriculture, and also discuss on C and N turnover of microbial biomass in Andisols. Microbial succession in acid soil has also not been investigated so much, but, some studies in another important acid soil, i.e. acid sulfate soil, were also reviewed briefly.

牛ふんコンポスト,豚ぷんコンポストおよび化学肥料を各種畑土壌(褐色低地土,黒ボク土および褐色森林土)にそれぞれ5年間連用し,栽培作物であるコマツナのZn・Cu吸収について調べた。その結果,以下のことが明らかとなった。1)コマツナのZn含有率は黒ボク土における豚ぷんコンポスト施用区の地上部および褐色森林土における豚ぷんコンポスト施用区の地上部および地下部で化学肥料区より高かった。コマツナのCu含有率はZnと異なり,家畜ふんコンポスト施用の影響は明確ではなかった。2)CuはZnに比べ作物移行性は低く,豚ぷんコンポストからいったん土壌に蓄積し,その後作物へ移行する画分が多いと考えられた。一方,Znはコンポストおよび土壌蓄積の双方から作物へ移行する傾向があると考えられた。3)コマツナの1株当たりのZn含有量は黒ボク土および褐色森林土の豚ぷんコンポスト施用区で化学肥料区と比べて高い値を示した。両元素ともにコマツナの含有量は細胞内の濃度よりもバイオマスに大きく依存しており,地上部に比べ地下部の含有量は著しく低かった。以上の結果より,豚ぷんコンポストは標準的な施用量であっても,長期間の連用によって褐色森林土のような可溶性重金属保持力が高い土壌においてZnの作物吸収が増大する可能性が示唆された。

緑肥エンバク野生種の栽培・すき込み後のトマト土壌病害の発生をピシウム苗立枯病,リゾクトニア苗立枯病,萎凋病,半身萎凋病および青枯病を対象に調査した結果,半身養凋病のような進行の遅い土壌病害の発生が抑制され,特に非殺菌時にその効果が顕著に認められた.その原因として,エンバク野生種栽培・すき込みに伴う土着の土壌微生物相の量的および質的変化による影響が,希釈平板法,微生物活性量およびPCR-DGGE法から示唆された.また,エンバク野生種由来の化学物質の抗菌性が関係している可能性も示した.