Kazunari Nagaoka

DGGE method for analyzing 16S rDNA of methanogenic archaeal community in paddy field soil.

A denaturing gradient gel electrophoresis (DGGE) method for analyzing 16S rDNA of methanogenic archaeal community in paddy field soil is presented. Five specific primers for 16S rDNA of methanogenic archaea, which were modified from the primers for archaea, were first evaluated by polymerase chain reaction and DGGE using genomic DNAs of 13 pure culture strains of methanogenic archaea. The DGGE analysis was possible with two primer pairs (0348aF-GC and 0691R; 0357F-GC and 0691R) of the five pairs tested although 16S rDNA of some non-methanogenic archaea was amplified with 0348aF-GC and 0691R. These two primer pairs were further evaluated for use in analysis of methanogenic archaeal community in Japanese paddy field soil. Good separation and quality of patterns were obtained in DGGE analysis with both primer pairs. A total of 41 DNA fragments were excised from the DGGE gels and their sequences were determined. All fragments belonged to methanogenic archaea. These results indicate that the procedure of DGGE analysis with the primer pair 0357F-GC and 0691R is suitable for investigating methanogenic archaeal community in paddy field soil.

Methanoculleus chikugoensis sp. nov., a novel methanogenic archaeon isolated from paddy field soil in Japan, and DNA-DNA hybridization among Methanoculleus species

A strictly anaerobic, irregularly coccoid, methanogenic archaeon, strain MG62T (= JCM 10825T = DSM 13459T), was isolated from paddy field soil in Chikugo, Fukuoka, Japan. The cells stained gram-negative, were 1.0-2.0 microm in diameter, were lysed by SDS and hypotonic solutions and were flagellated. Motility was not observed. The strain was able to use H2/CO2, 2-propanol/CO2, formate, 2-butanol/CO2 and cyclopentanol/CO2 as substrates for methanogenesis, but did not utilize acetate, ethanol, methanol or methylamines. The optimum temperature and pH were 25-30 degrees C and 6.7-7.2. Analysis of lipid component parts (core lipids, phospholipid polar head groups and glycolipid sugar moieties) showed the characteristic pattern of members of the family Methanomicrobiaceae except for the absence of glucose as a glycolipid sugar moiety. The G+C content of the DNA was 62.2 mol %. Sequence analysis of the 16S rDNA revealed that the strain belonged to the genus Methanoculleus. The strain had DNA-DNA hybridization values of less than 50% with type strains of Methanoculleus species. On the basis of phenotypic, genotypic and phylogenetic characteristics, the name Methanoculleus chikugoensis sp. nov. is proposed for strain MG62T (= JCM 10825T = DSM 13459T). The DNA hybridization study also revealed the close relationships of three species, Methanoculleus olentangyi, Methanoculleus bourgensis and Methanoculleus oldenburgensis, among Methanoculleus species.

Bacterial communities are more dependent on soil type than fertilizer type, but the reverse is true for fungal communities

Abstract The soil microbial community is strongly influenced by a wide variety of factors, such as soil characteristics and field management systems. In order to use biological indicators based on microbial community structure, it is very important to know whether or not these factors can be controlled. The present study aimed to determine whether soil type or fertilization has a greater influence on the soil microbial community based on denaturing gradient gel electrophoresis (DGGE) analysis of 12 experimental field plots containing four different soil types, Cumulic Andosol, Low-humic Andosol, Yellow Soil and Gray Lowland Soil, kept under three different fertilizer management systems since 2001 (the application of chemical fertilizer, the application of rice husk and cow manure, and the application of pig manure). Bacterial DGGE analysis using 16S rRNA genes and fungal DGGE analysis using 18S rRNA genes revealed that the bacterial community was related to the soil type more than the fertilization; however, the fungal community was related to the fertilization more than the soil type. These results might suggest that the fungal community is easier to control by fertilization than the bacterial community. Thus, we propose that indicators based on the fungal community might be more suitable as microbial indicators for soil quality.

Characterization of Cu-resistant bacterial communities in Cu-contaminated soils

Abstract Bacterial communities in a spoil heap in a copper mine, a forest soil with high Cu contamination, and an agricultural soil with low Cu contamination were characterized in terms of Gram-staining, plasmid frequency, pigmentation, Cu-resistance system, and predominant Cu-resistant bacterial species. Similarity existed in that the ratio of green colonies for sorbing Cu and Gram-negative bacteria increased with the increase of the Cu content of the medium regardless of the characteristics of the bacterial communities. It was found that the plasmid was not present in most of the Cu-resistant bacteria of the soils. Curesistant genera including Burkholderia, Alcaligenes, and Methylobacterium species were isolated from the Cu contaminated soils using YG agar plates treated with 2 mM Cu. Furthermore, the bacterium with the highest Cu resistance (MIC = 5.5 mm Cu) was identified as a Gram-positive bacterium Bacillus sp., though most of the Cu-resistant bacteria were Gram-negative.

A quantitative evaluation and phylogenetic characterization of oligotrophic denitrifying bacteria harbored in subsurface upland soil using improved culturability

This is the first investigation to show that oligotrophic denitrifying bacteria are dominant denitrifiers in subsurface upland soil. We examined the vertical distribution of denitrifying bacterial populations in upland soil using two kinds of enumeration media. The number of denitrifying bacteria, enumerated in subsurface soil layers by a 100-fold diluted nutrient broth (DNB) medium with NO3−, was two to three orders of magnitude greater than those enumerated by a conventional nutrient broth medium with NO3−, suggesting the dominance of oligotrophic denitrifying bacteria. Seventy-four percent of the total denitrifying bacterial isolates were DNB organisms of the oligotrophic type, which did not show appreciable growth on a conventional nutrient broth medium. The isolates were heterogeneous and were categorized as alpha (35 strains) and beta (19 strains) subdivisions of proteobacteria and high G+C gram-positive bacteria (7 strains) by 16S rRNA gene sequence analysis. The 35-alpha subdivision of proteobacterial isolates was of oligotrophic type and widely distributed from the surface to subsurface soil layers. Phylogenetic analysis indicated that some isolates belonged to groups with few or no cultivated representatives, and that one isolate may be a member of a new genus. This isolation procedure, using diluted media, is valuable in detecting diverse and novel denitrifying bacteria in the subsurface soil.

Combined Analyses of Bacterial, Fungal and Nematode Communities in Andosolic Agricultural Soils in Japan

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.

Soil properties affecting phosphorus forms and phosphatase activities in Japanese forest soils: Soil microorganisms may be limited by phosphorus

Abstract To explore a possible phosphorus limitation of soil microbial processes, we fractionated phosphorus in Japanese forest soils (10 Inceptisols, three allophanic Andisols, seven nonallophanic Andisols, and one Spodosol) by sequential extraction into inorganic P (Pi) and organic P (Po) in H2O, 0.5 M NaHCO3, 0.1 M NaOH, 1 M HCl and conc. HCl fractions, and total P in residual fractions. NaOH-Pi and NaOH-Po fractions were the largest P components in all soil types. Apart from H2O-Pi, NaOH-Pi, and NaOH-Po, P concentration in each fraction did not differ significantly among soil types. Concentrations of P in fractions H2O-Pi, H2O-Po, 0.1 M NaOH-Pi, 0.1 M NaOH-Po, and residual P were correlated with active Al, but not with active Fe, indicating a more significant contribution of Al in controlling P forms in the soils. The proportion of available P (H2O + NaHCO3 − P) to total P was negatively affected by active Al and Fe contents and by pH in the soils. High phosphomonoesterase and phosphodiesterase are known to be indicators of low soil P availability, and both activities were higher in soils with low available P in this study, suggesting that microorganisms of these forest soils may be P limited.

Plant roots influence microbial activities as well as cadmium and zinc fractions in metal-contaminated soil

Changes in soil pH, soil heavy metal forms, and the metabolic diversity of microbial communities were examined in soil samples collected in 1-mm increments from barley roots in soil contaminated with cadmium (Cd) and zinc (Zn) using a rhizobox system. Concentrations of exchangeable Cd and Zn increased near the roots owing to a decrease in soil pH. Conversely, the concentration of inorganically bound Cd and Zn decreased near the roots. Despite having the highest concentration of the most toxic exchangeable metals, the rhizosphere also had the highest bacterial and fungal metabolic activity and diversity when assessed using BIOLOG plates. Therefore, the promoting effects of root exudates on microbial activity could outweigh the adverse effects of Cd and Zn on microorganisms in the rhizosphere.

Identification of the gene for disaggregatase from Methanosarcina mazei

The gene sequences encoding disaggregatase (Dag), the enzyme responsible for dispersion of cell aggregates of Methanosarcina mazei to single cells, were determined for three strains of M. mazei (S-6(T), LYC and TMA). The dag genes of the three strains were 3234 bp in length and had almost the same sequences with 97% amino acid sequence identities. Dag was predicted to comprise 1077 amino acid residues and to have a molecular mass of 120 kDa containing three repeats of the DNRLRE domain in the C terminus, which is specific to the genus Methanosarcina and may be responsible for structural organization and cell wall function. Recombinant Dag was overexpressed in Escherichia coli and preparations of the expressed protein exhibited enzymatic activity. The RT-PCR analysis showed that dag was transcribed to mRNA in M. mazei LYC and indicated that the gene was expressed in vivo. This is the first time the gene involved in the morphological change of Methanosarcina spp. from aggregate to single cells has been identified.

Trends of lettuce and carrot yields and soil enzyme activities during transition from conventional to organic farming in an Andosol

Abstract It has been reported that crop yields drop and then increase during the first few years of organic farming, and these yield recoveries have been attributed to gradual improvements in soil properties, such as soil microbial activities to mineralize nitrogen (N) or to suppress plant disease. To clarify whether yield increase during organic transition is caused by improvement of soil microbial activities, we compared identically managed organic and conventional plots of 1-year lettuce (Lactuca sativa L.)–carrot (Daucus carota L.) rotation for 3 years (organic plots: first 3 years after switching from conventional to organic management; conventional plots: managed in the same way as organic plots for 3 years but receiving chemical fertilizer, fungicide, insecticide and herbicide) in an Andosol field. During organic transition, yields of organic lettuce and carrots were lower than those of conventional lettuce and carrots for only the first year. Yield drop and recovery of lettuce were thought to be caused by changes in the amount of N uptake, though yield fluctuation of carrots was mainly caused by damage from insects. Although soil enzyme activities may be responsible for N mineralization, various soil enzyme activities promptly responded to organic amendment to become higher under organic management than under conventional management even after the first lettuce cropping (6 months after switching to organic management; much shorter than the period of organic transition). However, discriminant analysis using activities of six soil enzymes (dehydrogenase, β-glucosidase, β-galactosidase, α-glucosidase, cellulase and protease) indicated that 18–24 months (a period close to that of the organic transition) were needed for the pattern of various soil enzyme activities to be in a steady state after switching to organic management. The pattern of soil enzyme activities fluctuating to a plateau during the second lettuce cropping seemed to show a tendency similar to that of N uptake and yield of lettuce during organic transition. Soil available N in organic plots also became higher than that in conventional plots in the third year. These results suggested that improved N uptake and yield of lettuce during organic transition in an Andosol might be caused by either improvement in various soil enzyme activities or accumulation of soil available N. Yield response of carrots demanding less N was attributed not to N mineralization but to damage from insects.