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Objective: PCR-DGGE was used to examine the bacterial communities associated with sclerotia of Sclerotium rolfsii, the causal agent of soybean stem rot. Methodology and Results: Fungal sclerotia were buried in soil amended with organic matter and incubated under flooded conditions for 15 or 30 days in a greenhouse and in the field. The viability and associated bacterial communities of the recovered sclerotia were investigated. In samples from soil amended with rice straw or wheat bran and flooded for 30 days, DGGE band patterns revealed the greatest bacterial diversity. In the greenhouse, for example, the diversity index from organic amendment under 30 day flooding was greater than 1.80, whereas the index for other treatments was less than 1.5. In the field experiment, this pattern was similar. The diversity index derived from DGGE band patterns had a negative relationship with sclerotial viability or disease incidence. The nucleotide sequences of the DGGE bands revealed that members of the Clostridiaceae were dominant in samples that had been flooded for 30 days, whereas Oxalobacteraceae, Nocardiaceae, and Actinomycetaceae were major groups under unflooded conditions. Conclusion: This is the first report of a soil bacterial flora associated with S. rolfsii sclerotia under flooded conditions.

Fungi play an important role in the degradation of biodegradable plastics (BPs) in soil. However, little is known about their dynamics in the soil during the degradation of BPs. We studied the community dynamics of BP-degrading fungi during poly(butylene succinate-co-adipate) (PBSA) film degradation in two different types of soils using culture-dependent and culture-independent methods. The Fluvisol and the Andosol soils degrade embedded PBSA films at high and low speeds, respectively. The number of PBSA emulsion-degrading fungi that increased in the Fluvisol soil was higher than that in the Andosol soil after embedding with PBSA films. We succeeded in detecting internal transcribed spacer 1 (ITS1) regions those matched that of the fungi by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) in both soils. Our results suggest that fungal community analyses using PCR-DGGE in combination with BP degraders isolation techniques enables the monitoring of BP films-degrading fungi. Monitoring fungal population dynamics during the degradation of films comprising poly(butylene succinate-co-adipate) in a Fluvisol and an Andosol soil.

Fungi play an important role in the degradation of biodegradable plastics (BPs) in soil. However, little is known about their dynamics in the soil during the degradation of BPs. We studied the community dynamics of BP-degrading fungi during poly(butylene succinate

Crop residues are produced from agriculture in large amounts globally. Crop residues are known to be a source of nitrous oxide (N

Nitrogen fertilizer and crop residue are important sources of N2O emission in agricultural ecosystems. This study aims to investigate N2O emission and microbial N2O production process after potato crop residue input and fertilizer application to an Andosol field. The experiment comprised crop residue treatments as follows: with residue (WR) and without residue (NR). We measured N2O emission, N2O isotopomer ratios (bulk N isotope ratios and intramolecular 15N site preference), and environmental parameters to estimate microbial N2O production processes. Comparative analysis of N2O isotopomer ratios and environmental parameters suggested that nitrification and bacterial denitrification were main N2O production processes after N application. Nitrifier denitrification would also contribute to N2O production after N application. In contrast, bacterial denitrification and fungal denitrification were important N2O production processes in the WR treatment after crop residue input, while nitrification also contributed to N2O production. Fungal denitrification was possibly important process during N2O peak after crop residue input. These findings reveal that real-time measurements of N2O isotopomers performed using a quantum cascade laser spectrometry system are useful for estimating microbial N2O production on a field scale.

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 ...

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 relatio...

Andosols comprise one of the most important soil groups for agricultural activities in Japan because they cover about 46.5% of arable upland fields. In this soil group, available phosphorus (P) is accumulated by application of excessive fertilizer, but little is known about the influence of increasing P availability on microbial community diversity at large scales. We collected soil samples from 9 agro-geographical sites with Andosol soils across an available P gradient (2048.1–59.1 mg P2O5·kg−1) to examine the influence of P availability on the fungal community diversity. We used polymerase chain reaction – denaturing gradient gel electrophoresis to analyze the fungal communities based on 18S rRNA genes. Statistical analyses revealed a high negative correlation between available P and fungal diversity (H′). Fungal diversity across all sites exhibited a significant hump-shaped relationship with available P (R2 = 0.38, P < 0.001). In addition, the composition of the fungal community was strongly correlated with the available P gradient. The ribotype F6, which was positively correlated with available P, was closely related to Mortierella. The results show that both the diversity and the composition of the fungal community were influenced by available P concentrations in Andosols, at a large scale. This represents an important step toward understanding the processes responsible for the maintenance of fungal diversity in Andosolic soils.

Soybean hosts the symbiotic nitrogen-fixing soil bacterium Bradyrhizobium japonicum, that can produce the greenhouse gas nitrous oxide. This study shows that nitrous oxide emissions from soybean ecosystems can be biologically mitigated at a field scale by inoculation with strains of B. japonicum that have increased nitrous oxide reductase activity.

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.

We simultaneously examined the bacteria, fungi and nematode communities in Andosols from four agro-geographical sites in Japan using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and statistical analyses to test the effects of environmental factors including soil properties on these communities depending on geographical sites. Statistical analyses such as Principal component analysis (PCA) and Redundancy analysis (RDA) revealed that the compositions of the three soil biota communities were strongly affected by geographical sites, which were in turn strongly associated with soil characteristics such as total C (TC), total N (TN), C/N ratio and annual mean soil temperature (ST). In particular, the TC, TN and C/N ratio had stronger effects on bacterial and fungal communities than on the nematode community. Additionally, two-way cluster analysis using the combined DGGE profile also indicated that all soil samples were classified into four clusters corresponding to the four sites, showing high site specificity of soil samples, and all DNA bands were classified into four clusters, showing the coexistence of specific DGGE bands of bacteria, fungi and nematodes in Andosol fields. The results of this study suggest that geography relative to soil properties has a simultaneous impact on soil microbial and nematode community compositions. This is the first combined profile analysis of bacteria, fungi and nematodes at different sites with agricultural Andosols.

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.

Despite their close association with human activities, plant pathogenic fungi have rarely been found in archaeological excavations. We report here that a fungus was closely associated with human activities even in prehistoric times. Sclerotium-like objects were found at historical sites (4000 to 400 BP) on the island of Hokkaido, northern Japan. They were spherical, 0.3-1.0 mm in diameter, and had a medulla and rind. Some had leaf fragments on the surface or a protuberance that resembled emerging sporocarp primordia. These traits indicated that they were sclerotia of the snow mold fungus, Typhula ishikariensis biotype B.

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...

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 h...

Objective : The bacterial communities concomitant with sclerotia of Sclerotium rolfsii , the causal agent of soybean stem rot, were examined by using PCR-DGGE. Methodology and results : Fungal sclerotia were buried in soil amended with organic matter and incubated under flooded conditions for 15 or 30 days in a greenhouse and in the field. The recovered sclerotia were examined for their viability and their concomitant bacterial communities. The DGGE band patterns showed the largest bacterial diversity in samples from soil amended with rice straw or wheat bran and flooded for 30 days. In the greenhouse for instance, the diversity index from organic amendment under 30 day flooding was higher than 1.80, while for other treatments the index was less than 1.5. This trend was similar in the field experiment. There was a negative relationship between the diversity index from DGGE band patterns and sclerotial viability or disease incidence. Examination of the nucleotide sequences of the DGGE bands revealed that members of Clostridiaceae were dominant in the samples flooded for 30 days, whereas Oxalobacteraceae, Nocardiaceae, and Actinomycetaceae were major groups under unflooded conditions. Conclusion : This is the first report of the soil bacterial flora concomitant with sclerotia of S. rolfsii under flooded conditions. Keywords : bacterial community, diversity, PCR-DGGE, Sclerotium rolfsii , sequencing, soybean

An oligonucleotide probe targeting the rRNA of Erwinia herbicola and Erwinia ananas was designed to detect their cells using fluorescence in situ hybridization. The Cy3-labeled probe hybridized strongly with these species but very weakly with nontarget species such as Erwinia mallotivora, Erwinia nigrifluens, Erwinia cypripedii, Erwinia chrysanthemi, Erwinia carotovora subsp. carotovora, E. carotovora subsp. atroseptica, and Escherichia coli. This technique visualized E. herbicola cells after inoculation of kumquat fruits. The probe is promising as a tool for studying population dynamics of E. herbicola and E. ananas.

There is an increasing interest in the extraction of DNA from soil, since molecular techniques facilitate the analysis of the microbial community including unculturable microorganisms. Though various methods of direct DNA extraction have been devised, it still remains difficult to extract DNA from some soils. We developed an improved method to extract DNA from such "recalcitrant soils". And-isol, volcanic ash soils are recalcitrant in this context. Skim milk increased the efficacy of DNA extraction from such soils, when added to the extraction buffer as an adsorption competitor to soil particles. This method is applicable to molecular community analysis of soils which strongly adsorb DNA.

DNA extraction has been difficult from some types of soil. Seven soil samples from agricultural fields and a forest, which were mainly volcanic ash soils, were used. Soil DNA could be extracted from only two of them using a commercially available kit exploiting bead-beating. When skim milk was added to the extraction buffer at 40 mg g-1 soil, DNA could be detected by electrophoresis from all the samples, indicating that the DNA from lysed cells was adsorbed by soil colloids. The addition of skim milk did not affect PCR-DGGE profiles. The improved method is applicable to the analysis of molecular communities in soils which strongly adsorb DNA.

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.

Fungi play important and diverse roles in soil ecosystems. They act as plant pathogens, mycorrhizal symbionts and most importantly, as the principal decomposers of organic materials (Christensen, 1989; Thorn, 1997). Fungi also represent a dominant component of the soil microflora in terms of biomass (Thorn, 1997). Compared with bacterial communities, however, knowledge regarding the diversity and functions of soil fungal communities remains limited. Culture-independent molecular techniques, comprising of direct DNA extraction from soil followed by PCR and electrophoresis or cloning, have been introduced to investigate soil fungal communities (Anderson & Cairney, 2004). These techniques facilitate the detection of fungi, including fastidious or non-culturable strains, and an understanding of the fungal community structures and dynamics in soil (Hoshino & Matsumoto, 2007; Vandenkoornhuyse et al., 2002). Molecular techniques have provided novel insights and significant advances in research on soil fungal ecology and have been applied to various soils in different ecosystems, such as forests (Perkiomaki et al., 2003), grasslands (Brodie et al., 2003), dunes (Kowalchuk et al., 1997), stream sediments (Nikolcheva et al., 2003) and agricultural fields (Gomes et al., 2003). For example, in agricultural soils, a fungal community is affected by plant growth (Gomes et al., 2003) and cultural practices, such as application of fertilizers and pesticides (Girvan et al., 2004). With the development of new technologies, accumulating molecular data has contributed to the establishment of database combined with other environmental data and facilitated metaanalysis on a large scale. In agricultural soils, fungal communities are directly and indirectly related to crop production. Technological advances in molecular methods would help elucidate such a complicated relationship. Here I present molecular techniques applied to soil fungal community analyses, particularly in agricultural soils and discuss their limitations and future applications.

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.