Yuko Takada Hoshino

Comparison of 18S rDNA primers for estimating fungal diversity in agricultural soils using polymerase chain reaction-denaturing gradient gel electrophoresis

Abstract Direct DNA extraction followed by 18S rDNA polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) is widely used for the analysis of fungal diversity in agricultural soil ecosystems. Various PCR primer sets have been used for fungi, but few reports have compared the properties of the primers. We investigated the properties of four widely used primer sets for fungal 18S rDNA DGGE: (1) NS1/GCFung, (2) FF390/FR1(N)-GC, (3) NS1/FR1(N)-GC (for single PCR) and (4) NS1/EF3 for the first PCR and NS1/FR1(N)-GC for the second PCR (for nested PCR). Using six soil samples from upland and paddy fields in Japan, the primers were compared in terms of PCR amplification efficacy, detection and reproducibility of the DGGE banding profiles, the obtained diversity indices, and the discrimination ability of the fungal communities using DGGE. The efficacy of PCR amplification using primer set 1 and the first PCR of primer set 4 was better than that using primer sets 2 and 3. In DGGE analysis, the PCR products of primer sets 3 and 4 showed the highest diversity indices. However, these primer sets had drawbacks, namely, the presence of non-specific aggregates and poor reproducibility of the DGGE profiles. Although primer sets 1 and 2 yielded shorter sequences of similar length, the PCR products with primer set 1 showed higher diversity indices than those with primer set 2. Multidimensional scaling analysis of the DGGE profiles indicated that primer set 1 could most clearly discriminate each fungal community in the soil samples. Although each primer set had advantages and disadvantages, together our analyses indicated that primer set 1 is the most suitable for detecting fungal diversities in soil using DGGE analysis. Our results are useful for selecting primers according to the aim of a particular study.

Changes in fungal community structure in bulk soil and spinach rhizosphere soil after chemical fumigation as revealed by 18S rDNA PCR-DGGE

Abstract To analyze the impact of chloropicrin and 1,3-dichloropropene on fungal community structure in bulk soil and spinach rhizosphere soil in a field, developed a new nested polymerase chain reaction (PCR) method to facilitate the detection of major fungal taxa and we used the method to monitor 18S rDNA PCR-denaturing gradient gel electrophoresis (DGGE) profiles for 3 years. The cropping system consisted of soil fumigation in September and two subsequent consecutive spinach cultivations each year. The soil was treated with fumigants (chloropicrin at 20 mL m−2 or 1,3-dichloropropene at 32 mL m−2) and covered with polyethylene film for approximately 2 weeks. The nested PCR method with primer pairs AU2/AU4 and GC-FR1/FF390 successfully amplified sequences from all major fungal taxa, but not from plants or oomycetes, which were amplified from rhizosphere soil samples using direct PCR with the primer pair GC-FR1/FF390. The DGGE analysis of the nested PCR products indicated that the chloropicrin treatment had a greater impact on the fungal community than 1,3-dichloropropene, both in terms of the magnitude and duration of the effect. Chloropicrin treatment changed DGGE profiles drastically and reduced the diversity index H′ in both bulk soil and rhizosphere soil 2 months after fumigation. Profiles and diversity indices did not recover completely after 1 year. Bands with high sequence similarity to ascomycetous fungi decreased in intensity and, conversely, bands inferred to represent chytridiomycota became dominant. In contrast, 1,3-dichloropropene did not reduce the diversity index significantly after 2 months. The DGGE profiles of 1,3-dichloropropene plots revealed a smaller change 2 months after fumigation, but became indistinguishable from those of control plots after 6 months. Spinach cultivation also affected the soil fungal community structure because the differences in DGGE profiles between control and chloropicrin plots were smaller in rhizosphere soil than in bulk soil 2 months after fumigation. These results suggest that the rhizosphere effect may contribute to minimizing the effect of chemical fumigation.

Complete genome sequence of Bradyrhizobium sp. S23321: insights into symbiosis evolution in soil oligotrophs.

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.

Comparison among amoA Primers Suited for Quantification and Diversity Analyses of Ammonia-Oxidizing Bacteria in Soil

Ammonia monooxygenase subunit A gene (amoA) is frequently used as a functional gene marker for diversity analysis of ammonia-oxidizing bacteria (AOB). To select a suitable amoA primer for real-time PCR and PCR-denaturing gradient gel electrophoresis (DGGE), three reverse primers (degenerate primer amoA-2R; non-degenerate primers amoA-2R-GG and amoA-2IR) were examined. No significant differences were observed among the three primers in terms of quantitative values of amoA from environmental samples using real-time PCR. We found that PCR-DGGE analysis with the amoA-2IR primer gave the best results in this studied soil. These results indicate that amoA-2IR is a suitable primer for community analysis of AOB in the environment.

Mitigation of soil N2O emission by inoculation with a mixed culture of indigenous Bradyrhizobium diazoefficiens

Agricultural soil is the largest source of nitrous oxide (N2O), a greenhouse gas. Soybean is an important leguminous crop worldwide. Soybean hosts symbiotic nitrogen-fixing soil bacteria (rhizobia) in root nodules. In soybean ecosystems, N2O emissions often increase during decomposition of the root nodules. Our previous study showed that N2O reductase can be used to mitigate N2O emission from soybean fields during nodule decomposition by inoculation with nosZ++ strains [mutants with increased N2O reductase (N2OR) activity] of Bradyrhizobium diazoefficiens. Here, we show that N2O emission can be reduced at the field scale by inoculation with a mixed culture of indigenous nosZ+ strains of B. diazoefficiens USDA110 group isolated from Japanese agricultural fields. Our results also suggested that nodule nitrogen is the main source of N2O production during nodule decomposition. Isolating nosZ+ strains from local soybean fields would be more applicable and feasible for many soybean-producing countries than generating mutants.

Relationships between ammonia oxidizers and N2O and CH4 fluxes in agricultural fields with different soil types

Abstract Nitrous oxide (N2O) is a greenhouse gas that contributes to the destruction of stratospheric ozone, and agricultural soil is an important source of N2O. Aerobic soils are sinks for atmospheric methane (CH4), a greenhouse gas. Ammonia monooxygenase (AMO) can oxidize CH4, but CH4 is mostly oxidized by methane monooxygenase (MMO), and CH4 oxidation by AMO is generally negligible in the soil. We monitored the N2O and CH4 fluxes after urea application in fields containing different soils using an automated sampling system to determine the effects of environmental and microbial factors on the N2O and CH4 fluxes. The soil types were Low-humic Andosol (Gleyic Haplic Andosol), yellow soil (Gleyic Haplic Alisol) and gray lowland soil (Entric Fluvisol). Cumulative N2O emissions from the yellow soil were higher than those from other soil types, although the difference was not significant. The CH4 uptake level by Andosol was one order of magnitude higher than that by other soils. There were significant relationships between the ammonia oxidation potential, AOB and AOA amoA copy numbers, and the CH4 uptake. In contrast, the gene copy numbers of methane-oxidizing bacteria (MOB) pmoA were below the detection limit. Our results suggested that the AMOs of AOB and AOA may have more important roles than those previously considered during CH4 oxidation in agricultural soils treated with N fertilizers.

Effect of dicyandiamide and polymer coated urea applications on N2O, NO and CH4 fluxes from Andosol and Fluvisol fields

Abstract Soil type influences the effectiveness of enhanced-efficiency fertilizers in reducing nitrous oxide (N2O) and nitric oxide (NO) emissions, although the effect has not been well studied. We measured N2O, NO and methane (CH4) fluxes after the application of enhanced-efficiency fertilizers and conventional fertilizer (urea) in two contrasting soils, an Andosol and a Fluvisol, in lysimeter fields. Brassica rapa var. perviridis L.H.Bailey (komatsuna) was cultivated for 1.5 months in spring and in autumn. A nitrification inhibitor, dicyandiamide (DCD), and polymer coated urea (CU) were tested in the spring and autumn experiments, respectively. In spring, DCD was effective in reducing N2O and NO emissions in the Andosol but not in the Fluvisol, compared with urea. Nitrification was likely to be a more important production process for N2O and NO in the Andosol than in the Fluvisol. This difference in N2O and NO production processes was inferred to be the main reason why DCD effectively reduced N2O and NO emissions only in the Andosol. Yield-scaled N2O emission for DCD was lower by 63% than for urea in the Andosol, but no difference was observed in the Fluvisol. In autumn in the Andosol, CU increased N2O emission compared with urea, but no difference was observed for NO emissions. In the Fluvisol, CU was not effective in reducing N2O and NO emissions. CH4 uptake from the Andosol was significantly higher than that from the Fluvisol. Fertilizer type had no effect on cumulative CH4 uptake in either soil. Our results showed that the effectiveness of DCD and CU in reducing N2O and NO emissions varied with soil because the main production processes of these gases varied with soil.

Magnetospinography visualizes electrophysiological activity in the cervical spinal cord

Diagnosis of nervous system disease is greatly aided by functional assessments and imaging techniques that localize neural activity abnormalities. Electrophysiological methods are helpful but often insufficient to locate neural lesions precisely. One proposed noninvasive alternative is magnetoneurography (MNG); we have developed MNG of the spinal cord (magnetospinography, MSG). Using a 120-channel superconducting quantum interference device biomagnetometer system in a magnetically shielded room, cervical spinal cord evoked magnetic fields (SCEFs) were recorded after stimulation of the lower thoracic cord in healthy subjects and a patient with cervical spondylotic myelopathy and after median nerve stimulation in healthy subjects. Electrophysiological activities in the spinal cord were reconstructed from SCEFs and visualized by a spatial filter, a recursive null-steering beamformer. Here, we show for the first time that MSG with high spatial and temporal resolution can be used to map electrophysiological activities in the cervical spinal cord and spinal nerve.

O-1-13. Diagnosis of lumbar radiculopathy using magnetospinography

Introduction We previously reported noninvasive visualization of neural activities in the lumbar spine using magnetospinography (MSG). We report here MSG findings of a patient with lumbar radiculopathy. Methods Using a newly developed superconducting quantum interference device (SQUID) magnetometer, we measured the neuromagnetic fields of a patient with L4/-5 disc herniation at the surface of the lumbar spine area after stimulation of the peroneal nerves at the fibular head. Current sources were estimated using spatial filter techniques and were superimposed on X-ray images of the lumbar spine. Results and discussion The action currents calculated from MSG showed a distribution pattern of axonal activity with an intra-axonal current and depolarization/repolarization current. On the contralateral healthy side, the nerve action current passed through the L5 intervertebral foramen and propagated in the caudal-to-cranial direction. On the affected side, the nerve action current attenuated at the L4/-5 intervertebral disc level. Thus, noninvasive surface MSG visualizes propagating spinal nerve excitation in response to peroneal nerve stimulation with high spatial and temporal resolution, allowing us to quantitatively evaluate the decreased action current in lumbar radiculopathy. MSG obtains a different perspective from MRI for the diagnosis of neuropathic pain and is expected to be a clinically useful modality.

T112. Visualization of electrical activities in the carpal tunnel area by magnetoneurography of median nerve

Introduction In recent years, diagnosis of the carpal tunnel syndrome is mainly performed by electrophysiological examination. In particular, the utility and reliability of inching method were demonstrated by some reports. However, electrophysiological examination is affected by the surrounding tissues such as bone and soft tissue, and the inching method is not technically easy and requires many electrodes. On the other hand, the magnetic field is not affected by the surrounding tissue. We have developed the magnetometer for the spinal cord and the spinal nerve activity and succeeded in measuring electrophysiological activity in the spinal cord and spinal nerve roots. Now, we are working on magnetoneurography (MNG) which is measurement of magnetic fields generated by the peripheral nerve. In this study, we aimed to evaluate spatiotemporal spread of digital nerve-evoked currents in the median nerve at the carpal tunnel area using MNG. Methods Using a newly developed superconducting quantum interference device (SQUID) magnetometer, neuromagnetic fields of 9 healthy volunteers’ hands were measured at the surface of the carpal tunnel area after stimulation of the digital nerves at the index or middle finger. Current sources were estimated using spatial filter techniques and were superimposed on X-ray images of the hand. We set a virtual electrode in the carpal tunnel along the median nerve. We reconstructed the current waveform from the magnetic field and calculated the nerve conduction velocity from the peak latency. We also measured sensory nerve action potential just above the carpal tunnel, 3 cm distal and 3 cm proximal to the carpal tunnel in response to stimulation of the digital nerves of the index finger. The conduction velocity was calculated from the peak latency. Results Neuromagnetic fields propagating from the finger to the wrist were successfully measured in all subjects. Distribution of action currents calculated from MNG showed the axonal activity pattern showing the intra-axonal current and the depolarization/ repolarization current. Nerve conduction velocity estimated from the MNG was 55.3 m/s and corresponded with the sensory nerve conduction velocity. Conclusion We could visualize action current at any point in carpal tunnel with high resolution using MNG. Moreover, MNG is not affected by the surrounding bone and soft tissues and can be fused with morphological images such as X-rays. MNG is expected to contribute to the clinical diagnosis and treatment of carpal tunnel syndrome.