Vertical Changes in Bacterial Communities in Percolating Water of a Japanese Paddy Field as Revealed by PCR-DGGE

Percolating water was sampled from the plow layer and subsoil layer in a Japanese paddy field, and the bacterial communities were compared together with floodwater by polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) targeting a partial 16S rRNA gene and subsequent sequencing. The number of DGGE bands ranged from 16 to 28 with no significant differences among the sampling sites and times. Only 2 bands were common for the three sources of water samples. DGGE bands specific for the floodwater samples and percolating water samples from the plow layer were identified, while percolating water samples from the subsoil layer did not show specific bands but displayed common bands to those of the floodwater samples (7 bands) and percolating water samples from the plow layer (1 band). Cluster analysis of the DGGE banding patterns showed a distinct clustering in the samples of percolating water from the plow layer and a closer relationship between the others. These results suggest that the bacterial communities in percolating water changed during downward movement through the plow layer and subsoil layer. Sequences of the DGGE bands specific for the samples of percolating water from the plow layer showed a close relationship with anaerobic bacteria such as iron-reducers or uncultured bacterial DNA isolated from environments that are considered to be less oxic. On the other hand, the sequences of the bands specific for the samples of floodwater and percolating water from the subsoil layer showed a close relationship with uncultured bacterial DNA isolated from freshwater environments.     

Unique viral capsid assembly protein gene (g20) of cyanophages in the floodwater of a Japanese paddy field

In order to evaluate the genetic diversity of cyanophage communities of rice fields, viral capsid assembly protein gene (g20) was amplified with primers CPS1 and CPS8. The DNA was extracted three times from viral concentrates obtained from floodwater samples collected in each of four different plots (no fertilizer; P and K chemical fertilizers; N, P, and K chemical fertilizers; and chemical fertilizers with compost). Denaturing gradient gel electrophoresis (DGGE) gave different g20 clones. The sequencing of DGGE bands revealed that the g20 genes of the floodwater were divergent and that the majority of clones formed several unique groups. However, they were more closely related to g20 sequences from freshwaters than to those from marine waters, suggesting that g20 genes in terrestrial aquatic environments are different from those in marine environments.     

Evaluation of rice–legume–rice cropping system on grain yield,nutrient uptake,nitrogen fixation,and chemical,physical, and biological properties of soil

To achieve higher yields and better soil quality under rice–legume–rice (RLR) rotation in a rainfed production system, we formulated integrated nutrient management (INM) comprised of Azospirillum (Azo), Rhizobium (Rh), and phosphate-solubilizing bacteria (PSB) with phosphate rock (PR), compost, and muriate of potash (MOP). Performance of bacterial bioinoculants was evaluated by determining grain yield, nitrogenase activity, uptake and balance of N, P, and Zn, changes in water stability and distribution of soil aggregates, soil organic C and pH, fungal/bacterial biomass C ratio, casting activities of earthworms, and bacterial community composition using denaturing gradient gel electrophoresis (DGGE) fingerprinting. The performance comparison was made against the prevailing farmers’ nutrient management practices [N/P₂O₅/K₂O at 40:20:20 kg ha⁻¹ for rice and 20:30:20 kg ha⁻¹ for legume as urea/single super-phosphate/MOP (urea/SSP/MOP)]. Cumulative grain yields of crops increased by 7–16% per RLR rotation and removal of N and P by six crops of 2 years rotation increased significantly (P < 0.05) in bacterial bioinoculants-based INM plots over that in compost alone or urea/SSP/MOP plots. Apparent loss of soil total N and P at 0–15 cm soil depth was minimum and apparent N gain at 15–30 cm depth was maximum in Azo/Rh plus PSB dual INM plots. Zinc uptake by rice crop and diethylenetriaminepentaacetate-extractable Zn content in soil increased significantly (P < 0.05) in bacterial bioinoculants-based INM plots compared to other nutrient management plots. Total organic C content in soil declined at 0–15 cm depth and increased at 15–30 cm depth in all nutrient management plots after a 2-year crop cycle; however, bacterial bioinoculants-based INM plots showed minimum loss and maximum gain of total organic C content in the corresponding soil depths. Water-stable aggregation and distribution of soil aggregates in 53–250- and 250–2,000 μm classes increased significantly (P < 0.05) in bacterial bioinoculants-based INM plots compared to other nutrient management plots. Fungal/bacterial biomass C ratio seems to be a more reliable indicator of C and N dynamics in acidic soils than total microbial biomass C. Compost alone or Azo/Rh plus PSB dual INM plots showed significantly (P < 0.05) higher numbers of earthworms’ casts compared to urea/SSP/MOP alone and bacterial bioinoculants with urea or SSP-applied plots. Hierarchical cluster analysis based on similarity matrix of DGGE profiles revealed changes in bacterial community composition in soils due to differences in nutrient management, and these changes were seen to occur according to the states of C and N dynamics in acidic soil under RLR rotation.     

Spatial changes of soil fungal and bacterial biomass from a sub-alpine coniferous forest to grassland in a humid, sub-tropical region

 Fungal and bacterial biomass were determined across a gradient from a forest to grassland in a sub-alpine region in central Taiwan. The respiration-inhibition and ergosterol methods for the evaluation of the microbial biomass were compared. Soil fungal and bacterial biomass both significantly decreased (P<0.05) with the shift of vegetation from forest to grassland. Fungal and bacterial respiration rates (evolved CO₂) were, respectively, 89.1 μl CO₂ g^–1 soil h^–1 and 55.1 μl CO₂ g^–1 soil h^–1 in the forest and 36.7 μl CO₂ g^–1 soil h^–1 and 35.7 μl CO₂ g^–1 soil h^–1 in the grassland surface soils (0–10 cm). The fungal ergosterol content in the surface soil decreased from the forest zone (108 μg g^–1) to the grassland zone (15.9 μg g^–1). A good correlation (R ²=0.90) was exhibited between the soil fungal ergosterol content and soil fungal CO₂ production (respiration) for all sampling sites. For the forest and grassland soil profiles, microbial biomass (respiration and ergosterol) declined dramatically with depth, ten- to 100-fold from the surface organic horizon to the deepest mineral horizon. With respect to fungal to bacterial ratios for the surface soil (0–10 cm), the forest zone had a significantly (P<0.05) higher ratio (1.65) than the grassland zone (1.05). However, there was no fungal to bacterial ratio trend from the surface horizon to the deeper mineral horizons of the soil profiles. Received: 30 March 2000     

Molecular identification of methane oxidizing bacteria in a Japanese rice field soil

By using cultivation-independent techniques, community changes of methane-oxidizing bacteria (MOB) in rice bulk soils were investigated under field conditions in a Japanese rice field. The representative soil samples were collected during the typical rice growing season and nonrice growing period all year round. Statistical characterization of denaturing gradient gel electrophoresis (DGGE) community patterns of MOB pmoA/amoA functional gene fragments showed that MOB community structures in the rice bulk soils remained largely unchanged throughout the investigated period. The total intensity of six common DGGE bands that appeared consistently throughout the investigated period accounted for 64% of the total intensity of all 18 different DGGE bands detected. The low squared distance of the Ward cluster analysis of the DGGE pattern and the high Sorensen similarity coefficient (81%) also implied the high similarity of the MOB community structures. The stable MOB community structure did not couple well with the wide variation of soil water contents all year round. Sequencing analysis of the nine characteristic bands including six common bands revealed the presence of Type I, Type II methanotrophs, and β-proteobacterial ammonia oxidizers in rice bulk soils. In comparison with MOB type species, three DGGE bands showed a wide variation of the highly conserved amino acid residues, implying the presence of novel MOB bacteria inhabiting the rice bulk soil. The high diversity of MOB composition suggested that rice bulk soils might serve as an ideal reservoir for the dynamic changes of MOB in a rice field ecosystem in response to environment changes.     

Comparison of Bacterial Communities Associated with Microcrustaceans from the Floodwater of a Paddy Field Microcosm: Estimation Based on DGGE Pattern and Sequence Analyses

Bacterial communities associated with five kinds of microcrustaceans ( Tanycypris sp., Moina sp., Mesocyclops sp., Cypretta sp. and Heterocypris sp.) from the floodwater of a paddy field microcosm were examined by the application of denaturing gradient gel electrophoresis (DGGE) to PCR-amplified 16S rDNA products with universal bacterial primers and by sequencing of characteristic DGGE bands. The number of DGGE bands of the associated bacteria was small, indicating the association of specific bacterial members with the microcrustaceans studied, among which Tanycypris sp. showed the smallest number of bands. Principal component analysis (PCA) demonstrated that the community structure of the associated bacteria could be divided into three groups: Podocopida ( Tanycypris sp., Cypretta sp. and Heterocypris sp.), Moina sp. and Mesocyclops sp., and further analysis separated Tanycypris sp. and Heterocypris sp. into different clusters. The duration of the incubation period affected the bacteria associated with Tanycypris sp., Moina sp. and Cypretta sp. only. Nearly all of the associated bacteria belonged to Gram-negative bacteria, especially the Cytophaga-Flavobacterium-Bacteroides (CFB) group. Closest relatives of the DGGE bands common to three Podocopida and Mesocyclops sp. belonged to an invertebrate endosymbiont.     

Bacterial communities associated with nodal roots of rice plants along with the growth stages: Estimation by PCR-DGGE and sequence analyses

Bacterial communities in rice roots that developed from different nodes and at different growth stages were compared by using polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE) analysis of 16S rDNA. Rice root samples were collected at three stages, namely tillering (July 2), maximum tillering (July 21), and ripening (September 12). The bacterial diversity in rice roots was found to increase along with the growth stages of the rice plants as well as the root age from the numbers of DGGE bands. The community structure of the bacteria was also found to change with the growth stages and root age from cluster analysis. Sequence analysis of the DGGE bands indicated that the dominant bacteria associated with rice roots were Gram-negative bacteria, especially β-Proteobacteria irrespective of the growth stages and root age. DGGE bands related to Janthinobacterium agaricidamnosum W1r3T and Clostridium sp. FCB90-3 were ubiquitous in many roots irrespective to the sampling date. Principal component analysis enabled to characterize the DGGE bands related to nitrogen-fixing Azoarcus spp., and Azovibrio sp. BS20-3 in the samples collected on July 2 and on July 21, and the myxobacteria collected on September 12, respectively, as representative bacteria in the bacterial communities. The habitat around older rice roots at every sampling date was more reductive than that around younger rice roots, and the DGGE bands related to Spirochaeta spp. were specific in older roots at every sampling date. Some specific bacteria that were most closely related to the DGGE bands were found from principal component analysis to characterize young and old. roots at each growth stage as follows: aerobes Flavobacterium sp. 90 clone 2 and Janthinobacterium agaricidamnosus W1r3T in young roots and facultative anaerobes Dechloromonas sp. MissR and Anaeromyxobacter dehalogenans 2CP-3 in old nodal roots on July 2, strict anaerobe Geobacter pelophilus Dfr2 and aerobes Nitrosospira sp. Nsp17 and uncultured Nitrospira sp. clone 4-1 in old roots on July 21, and different Clostridium spp. in both young and old roots and Desulfovibrio magneticus RS-1 in old roots on September 12, respectively. A larger number of the closest relatives of anaerobic bacteria grew at the late stage than at the early stages, and in old roots than in younger roots. Thus, the environment of paddy roots was remarkably heterogeneous as a bacterial habitat, where not only the whole root system but also a root may create oxic and anoxic environments.     

Comparison of bacterial community structures at main habitats in paddy field ecosystem based on DGGE analysis

Bacterial communities at different habitats in a Japanese paddy field ecosystem were compared to understand the bacterial world in the ecosystem as a whole by analyzing data of the denaturing gradient gel electrophoresis (DGGE) band patterns and the sequenced DGGE bands. The habitats were floodwater, percolating water, microcrustacean inhabiting in floodwater, plow layer soil, rice roots, rice straw and rice straw compost incorporated in soil, rice straw placed on the soil surface, plant residues in paddy fields, and rice straw under composting process. Phylotype (band) richness, diversity, evenness, and stability of the bacterial communities at the respective habitats were evaluated based on the DGGE profile data. Phylotype richness was greater near plant residues, rice straw buried in soil and rice straw placed on soil surface, while it was smaller at microcrustacean and rice straw compost buried in soil. The samples from plow layer soil and rice straw compost buried in soil showed considerably higher index values for diversity, evenness, and stability, while those from rice straw placed on soil surface and microcrustacean had lower values of the indices than other habitats. Sequences of totally 250 DGGE bands were assigned to phyla or classes. Distribution of bacterial members to phylogenetic taxa was different among the respective habitats. Inhabitants in plow layer soil were most widely distributed among the groups (nine phyla: Proteobacteria, Chloroflexi, Chlorobi, Verrucomicrobia, Acidobacteria, Nitrospira, candidate division OP10, Cyanobacteria, and Actinobacteria), while those in floodwater and microcrustacean were restricted to only three phyla (Proteobacteria, Bacteroidetes, and Actinobacteria). Proteobacteria and Bacteroidetes were found at all the habitats and the habitats except for plow layer soil, respectively, whereas abundant members belonged to Chloroflexi and Actinobacteria in plow layer soil. “Comprehensive mapping” of DGGE fragments was conducted by principal component analysis based on evolutionary distances of the fragments to 202 reference bacterial strains to overview phylogenetic relationships of bacterial members among the respective habitats. The score plots with the first and second principal components distinctly characterized bacterial members at the respective habitats, and the similarity between the respective communities was clearly demonstrated. Overall, bacterial communities at the respective habitats were distinct and different in the diversity and stability to each other, which may have contributed to the diversity of overall bacterial communities in the paddy field ecosystem.     

DGGE fingerprinting of culturable soil bacterial communities complements culture-independent analyses

Culture-dependent DGGE (CD DGGE) fingerprinting of the 16S rRNA gene was used to characterize mixed bacterial communities recovered on agar plates. Using R2A Agar as a growth medium, CD DGGE analysis resulted in clear banding patterns of sufficient complexity (16-32 major bands) and reproducibility to investigate differences in bacterial communities in a silt loam soil. Replicate CD DGGE profiles from plates inoculated with less-dilute samples (10⁻³) had a higher band count and were more similar (72-77%) than profiles from more-dilute samples (51-61%). Different culture media and incubation conditions resulted in distinct community fingerprints and increased the cumulative number of unique bands detected. When CD DGGE fingerprints were compared to profiles constructed from 16S rRNA genes obtained from culture-independent clone libraries (CB DGGE profiles) 34% of the bands were unique to the culture-dependent profiles, 32% were unique to the culture-independent profiles and 34% were found in both communities. These data demonstrate that culture-independent DGGE profiles are supplemented by the distinct bands detected in culture-dependent profiles. CD DGGE can be a useful technique to follow the dynamics of distinct culturable fractions of the soil bacterial community.     

Microbial communities on litter of managed and abandoned alpine pastureland

Degradation of litter strongly depends on its chemical composition, which in turn affects the associated microorganisms. In the alpine region, the abandonment of pastures leads to a rigorous change in the composition of the litter layer, shifting from grass to highly recalcitrant dwarf shrub litter, thus affecting the food web and decomposition processes. Three species belonging to different functional groups (grasses, herbs, dwarf shrubs) and indigenous on managed and/or abandoned alpine pastureland were selected for this study, the annual grass Dactylis glomerata, characteristic for managed sites, Trollius europaeus as representative of a herb common on both managed and abandoned areas, and Vaccinium vitis-idea as typical dwarf shrub arising on abandoned alpine pastures. Litter bags containing litter material from either one of the three plant types were incubated on the soil surface for 9 weeks. DNA was extracted from the substrate, amplified and analysed using PCR–DGGE. Fingerprinting analyses of bacterial and fungal communities showed that the microbiota attached to the litter differed considerably depending on the plant type. Nonetheless, specific bacterial bands were present in the fingerprinting patterns of all three litter types. Identifying these organisms applying the 16S rRNA clone libraries revealed the dominance of bacteria from the class Bacteroidetes, representing two thirds of all identified band positions.     

Effects of option mitigating ammonia volatilization on CH4 and N2O emissions from a paddy field fertilized with anaerobically digested cattle slurry

A lysimeter experiment was carried out to evaluate the effects of the NH₃ volatilization mitigation by adding anaerobically digested cattle slurry (ADCS) alone, with wood vinegar (WV) or with a higher level of floodwater (HFW), on emissions of CH₄ and N₂O from a paddy soil planted with fodder rice. We have carried out the following treatments: (1) chemical fertilizer, (2) ADCS, (3) ADCS + WV, and (4) ADCS + HFW; the height of floodwater was 10 cm in the latter treatment, and it was 3 to 4 cm in the other treatments just before fertilizer applications. Nitrogen fertilizer rate added to soil in each treatment was 30 g NH₄⁺–N m⁻² (split in one basal and two top-dressing additions). Ammonia volatilization in the ADCS treatment was 2.7 g NH₃–N m⁻² throughout the growing season, and it was significantly reduced by 79% and 55% in the ADCS + WV and ADCS + HFW treatments, respectively. The total amount of CH₄ emitted in the ADCS treatment in the growing season was not significantly enhanced by the mitigation of NH₃ volatilization either by adding wood vinegar or by increasing the height of the floodwater. Negligible N₂O emissions were observed in all treatments during the growing period.     

Denaturing gradient gel electrophoresis analysis of gut bacterial community for the Japanese earthworms

The gut bacterial community structure for Pheretima hilgendorfi and P. heteropoda (Family Megascolecidae), and Allolobophora japonica (Family Lumbricidae) collected from agricultural grasslands in Japan was analyzed by denaturing gradient gel electrophoresis of polymerase chain reaction-amplified 16S rRNA gene fragments (PCR-DGGE) and compared with those in the surrounding soils. Denaturing gradient gel electrophoresis (DGGE) profiles indicated that each earthworm species had their own specific bacterial communities, and multidimentional scaling analysis grouped the DGGE profiles into three groups: gut samples from P. hilgendorfi and P. heteropoda, gut samples from A. japonica and samples from the surrounding soils. Nine dominant bands were identified by their direct sequencing and cloning. Major three bands from P. hilgendorfi and P. heteropoda were closely related to Bacillus species belonging to the phylum Firmicutes. Major four and two bands from A. japonica were closely related to the phyla Proteobacteria and Bacteroidetes, respectively.     

Effects of algae and fertilizer-nitrogen on pH, Eh and depth of aerobic soil in laboratory columns of a flooded sandy loam

 A sandy loam was incubated under floodwater in the laboratory either in the dark or in the light (7 h day, 20  °C; 17 h night, 15  °C) and with four N sources [control, ammonium carbonate [(NH₄)₂CO₃], ammonium chloride (NH₄Cl), potassium nitrate (KNO₃)]. In the dark, floodwater pH rose steadily from 6.4 to about 7.5 over 60 days in the control, KNO₃ and (NH₄)₂CO₃ treatments, but with NH₄Cl pH decreased to 5.8. In the light, algal growth began to affect the floodwater pH after 9 days. At the end of the night, pH values were similar for all treatments to those kept in the dark. During the day, pH changes depended on the morning pH value: the daily increase was zero at pH 5.6 rising to a maximum of about 2 units at pH 6.3 and falling again at higher pH values. Changes in the carbonate equilibria in response to CO₂ removal by algal photosynthesis partly explain the results, but increasing inputs of acid are also implicated below pH 6.3 possibly due to reduced volatilization and increased nitrification. Redox potential (Eh) in the floodwater was little affected by N treatment until algal growth began. Eh then decreased each day as pH rose and recovered during the night. The daily decrease in Eh per unit increase in pH rose from about 10 to 90 mV pH^–1 over the incubation period. Initially, therefore, O₂ concentration must have been increased during the day by algal photosynthesis (values <59 mV pH^–1), but later O₂ concentration must have fallen, due possibly to the decomposition of algal cells. The presence of algae initially increased the depth of the aerobic soil layer, but eventually an algal mat settled on the soil surface acting as a zone of O₂ demand as the algae decomposed. Received: 7 July 1997     

Transformations of nitrogen-15-labelled urea in a flooded soil as affected by floodwater algae and green manure in a growth chamber

 The effects of floodwater algae and green manure on transformations of ¹⁵N-urea were studied in columns of a sandy loam soil in a growth chamber. The columns were flooded and either kept in the light, to allow algal growth, or in the dark (control) for 17 days before adding the labelled urea. Changes in urea-, NO₃ ^–- and NH₄ ⁺-N levels and the pH of the floodwater were measured over the subsequent 41-day period, during which the control column remained in the dark and those containing algae were maintained either in the dark to cause the death of the algae or in the light. Volatilized NH₃ was monitored, and on termination of the experiment the distribution of ¹⁵N between NO₃ ^–, NH₄ ⁺ and organic forms was measured in the soil. Urea hydrolysis was most rapid in the presence of both living algae and green manure, followed by dead algae, and was slowest in the control. The concentration of NH₄ ⁺-N in the floodwater was, however, reduced in the presence of algae due to assimilation and NH₃ volatilization owing to the raised day-time pH in the floodwater. NH₃ volatilization for the first 10 days was rather high in the columns kept in the light compared to those in the dark. Total volatilization plus denitrification losses were greatest where dead algae were present, owing to the absence of live algae which assimilated more than half of the applied N. Algal growth in floodwater increased the depth of the aerobic soil layer present at the soil-water interface. Subsequently, under dark conditions, stimulated algal growth reduced the depth of the aerobic layer causing less nitrification, which resulted in lower losses of N due to denitrification, i.e. 17% of the applied urea-N as compared to 39% in the light treatments. Although the presence of green manure caused a marked increase in the rate of hydrolysis, algal assimilation prevented excessive N losses via volatilization, indicating that the retention of higher quantities of NH₄ ⁺-N may have increased fertilizer-N use efficiency. Received: 22 January 1999     

Technical experiences made with the litter bag test as required for the risk assessment of plant protection products in soil

Background, aim, and scope  The aim of this review was to assess the degree to which recent litter bag studies performed between 2002 and 2004 have followed the recommendations laid down in the ‘Effects of plant protection products on functional endpoints in soil (EPFES)’ guidance document and the Organisation for Economic Co-Operation and Development (OECD) Guidance Document on the Breakdown of Organic Matter in Litter bags (OECD, Guidance document on the breakdown of organic matter in litter bags, OECD series on testing and assessment, no 56, 36 pp, 2006), which can be required under European Union (EU) Directive 91/414/EEC (EU, Off J Eur Union, L230:1–32, 1991) for assessing the effect of plant protection products on non-target soil macro-organisms which contribute to the breakdown of organic matter. Materials and methods  The current review covers the analysis of key parameters of the litter bag method of a total of 29 litter bag studies conducted by six European Crop Protection Association companies. Results  The two validity criteria [i.e. 60% mass loss at the end of the study in the control and a maximum coefficient of variation of 40% for mass loss in the control plots (n = 6) during the first 6 months of the test] were met in all of the studies reviewed. In over half of the studies, more than 60% mass loss was reached in the control litter bags after about 6 months. Statistically significant effects on organic matter breakdown due to plant protection products were repeatedly observed during the first few months of the study conduct, including effects >10% up to >25% compared to the control. Discussion  The survey of 29 study reports—performed from May 2002 to May 2004—showed that litter bag studies are generally being performed following the ‘EPFES’ guidance document and the OECD guidance document no. 56. Transient, statistically significant effects >10% up to >25% compared to the control were determined repeatedly during the first few months of the study conduct, thus clearly indicating the sensitivity of the litter bag test system. Conclusions and perspectives  The litter bag study design is a valuable test system for the risk assessment of plant protection products on non-target soil macro-organisms which contribute to the breakdown of organic matter under EU Directive 91/414/EEC (EU, Off J Eur Union, L230:1–32, 1991). In general, it can be concluded that the litter bag test, as proposed by EPFES and the OECD guidance document, provides an integrative answer on the potential effects of plant protection products on organic matter breakdown in soil and can be continued to be used if the studies follow the recommendations as presented in the guidance documents closely.     

Bacterial communities in manganese nodules in rice field subsoils: Estimation using PCR-DGGE and sequencing analyses

Abstract

The phylogenetic positions of bacterial communities in manganese (Mn) nodules from subsoils of two Japanese rice fields were estimated using polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis followed by sequencing of 16S rDNA. The DGGE band patterns and sequencing analysis of characteristic DGGE bands revealed that the bacterial communities in Mn nodules were markedly different from those in the plow layer and subsoils. Three out of four common bands found in Mn nodules from two sites corresponded to Deltaproteobacteria and were characterized as sulfate-reducing and iron-reducing bacteria. The other DGGE bands of Mn nodules corresponded to sulfate and iron reducers (Deltaproteobacteria), methane-oxidizing bacteria (Gamma and Alphaproteobacteria), nitrite-oxidizing bacteria (Nitrospirae) and Actinobacteria. In addition, some DGGE bands of Mn nodules showed no clear affiliation to any known bacteria. The present study indicates that members involved in the reduction of Mn nodules dominate the bacterial communities in Mn nodules in rice field subsoils.     

Molecular analysis of β-proteobacterial ammonia oxidizer populations in surface layers of a submerged paddy soil microcosm

The structure of the β-proteobacterial autotrophic ammonia-oxidizing bacterial (AOB) communities in a microcosm of submerged paddy soil was determined by denaturing gradient gel electrophoresis (DGGE) analysis of 16S rRNA gene fragments amplified using AOB-selective primers. Shift in the community composition was observed 4 weeks after submergence. The communities from the surface layers (0–1, 2–3 mm) of the soil microcosm were different from those of the subsurface layers (6–9, > 15 mm) and DGGE bands specific to each layer were detected. The majority of the retrieved sequences were Nitrosospira-like, whereas no Nitrosomonas-like sequences were obtained. The 16S rDNA primer set also amplified sequences that were not related to the known Nitrosospira-Nitrosomonas group, although they showed a close relationship with other groups of β-proteobacteria. The results suggest that Nitrosospira-like populations are dominant AOB populations in the submerged paddy soil, and that the oxic layer of submerged paddy soil harbours the specific AOB.     

Dynamics and stratification of bacteria and fungi in the organic layers of a scots pine forest soil

The abundance and micro-stratification of bacteria and fungi inhabiting the organic layers of a Scots pine forest (Pinus sylvestris L.) were investigated. An experiment using stratified litterbags, containing organic material of four degradation stages (fresh litter, litter, fragmented litter and humus) was performed over a period of 2.5 years. Dynamics and stratification of fluorescent stained bacteria and fungi, ratios between bacterial and fungal biomass, and relationships with moisture and temperature are described. Average bacterial counts in litter and fragmented litter were similar, i.e., approximately 5×10⁹ bacteriag^–1 (dry weight) organic matter, and significantly exceeded those in humus. The mean bacterial biomass ranged from 0.338 to 0.252mg carbon (C) g^–1 (dry weight) organic matter. Lengths of mycelia were significantly below the usually recorded amounts for comparable temperate coniferous forests. The highest average hyphal length, 53mg^–1 (dry weight) organic matter, was recorded in litter and decreased significantly with depth. The corresponding mean fungal biomass ranged from 0.050 to 0.009mg Cg^–1 (dry weight). The abundance of bacteria and fungi was influenced by water content, that of fungi also by temperature. A litterbag series with freshly fallen litter of standard quality, renewed bimonthly, revealed a clear seasonal pattern with microbial biomass peaks in winter. The mean hyphal length was 104mg^–1 (dry weight) and mean number of bacteria, 2.40×10⁹ bacteria g^–1 (dry weight). Comparable bacterial and fungal biomass C were found in the freshly fallen litter [0.154 and 0.132mgCg^–1 (dry weight) organic material, respectively]. The ratio of bacterial-to-fungal biomass C increased from 1.2 in fresh litter to 28.0 in humus. The results indicate the existence of an environmental stress factor affecting the abundance of fungi in the second phase of decomposition. High atmospheric nitrogen deposition is discussed as a prime factor to explain low fungal biomass and the relatively short lengths of fungal hyphae in some of the forest soil layers under study. Received: 26 June 1997     

Resistance gene analog polymorphisms (RGAPs) in wild emmer wheat (<Emphasis Type="Italic">Triticum dicoccoides</Emphasis>) and their ecological associations

Using the 8 specific primer pairs based on the conserved motifs of plant resistance genes, the plant disease resistance gene analog polymorphisms (RGAPs) in 15 wild emmer wheat (Triticum dicoccoides) populations from Israel had been detected. High genetic variations at the RGAP loci were observed in T. dicoccoides populations. A total of 254 discernible bands were obtained among 115 accessions, and 192 bands (75.6%) were polymorphic. Each genotype had a unique banding profile, and the genetic similarity coefficient ranged from 0.094 to 0.862. In T. dicoccoides, the proportion of polymorphic loci (P), the genetic diversity (He) and Shannon’s information index were 0.756, 0.362 and 0.541, respectively. The proportion of polymorphic loci (P) per population averaged 0.732 (range: 0.515–0.932); genetic diversity (He) averaged 0.271 (range: 0.212–0.338); and Shannon’s information index averaged 0.404 (range: 0.310–0.493). The coefficients of genetic distance (D) among populations averaged 0.107 (range: 0.043–0.178), and the results of Mantel test (r = 0.168, P = 0.091) showed that the estimates of genetic distance were geographically independent. Neighbor-joining cluster analysis suggested that the genetic relationships of T. dicoccoides populations were associated with their ecogeographic distribution. The hierarchical analysis of molecular variance (AMOVA) and the coefficient of gene differentiation (G _ST ) values revealed that most of the variations were presented within populations, although significant differences among populations and regions were also detected. The values of P and Shannon’s information index were negatively correlated with the two factors: Tdd (day–night temperature difference) and Ev (mean annual evaporation), whereas they were positively correlated with one water factor: Rn (mean annual rainfall). The correlation matrix between He in the RGAPs and geographic variables contained 20 significant (P < 0.05) correlations. The present study established that T. dicoccoides in Israel had a considerable amount of genetic variations at RGAP loci at least partly correlated with ecological factors.     

Microbial composition and diversity of an upland red soil under long-term fertilization treatments as revealed by culture-dependent and culture-independent approaches

Background, aim, and scope  Fertilization is an important agricultural practice for increasing crop yields. In order to maintain the soil sustainability, it is important to monitor the effects of fertilizer applications on the shifts of soil microorganisms, which control the cycling of many nutrients in the soil. Here, culture-dependent and culture-independent approaches were used to analyze the soil bacterial and fungal quantities and community structure under seven fertilization treatments, including Control, Manure, Return (harvested peanut straw was returned to the plot), and chemical fertilizers of NPK, NP, NK, and PK. The objective of this study was to examine the effects on soil microbial composition and diversity of long-term organic and chemical fertilizer regimes in a Chinese upland red soil. Materials and methods  Soil samples were collected from a long-term experiment station at Yingtan (28°15′N, 116°55′E), Jiangxi Province of China. The soil samples (0–20 cm) from four individual plots per treatment were collected. The total numbers of culturable bacteria and fungi were determined as colony forming units (CFUs) and selected colonies were identified on agar plates by dilution plate methods. Moreover, soil DNAs were extracted and bacterial 16S rRNA genes and fungal 18S rRNA genes were polymerase chain reaction amplified, and then analyzed by denaturing gradient gel electrophoresis (DGGE), cloning, and sequencing. Results  The organic fertilizers, especially manure, induced the least culturable bacterial CFUs, but the highest bacterial diversity ascertained by DGGE banding patterns. Chemical fertilizers, on the other hand, had less effect on the bacterial composition and diversity, with the NK treatment having the lowest CFUs. For the fungal community, the manure treatment had the largest CFUs but much fewer DGGE bands, also with the NK treatment having the lowest CFUs. The conventional identification of representative bacterial and fungal genera showed that long-term fertilization treatments resulted in differences in soil microbial composition and diversity. In particular, 42.4% of the identified bacterial isolates were classified into members of Arthrobacter. For fungi, Aspergillus, Penicillium, and Mucor were the most prevalent three genera, which accounted for 46.6% of the total identified fungi. The long-term fertilization treatments resulted in different bacterial and fungal compositions ascertained by the culture-dependent and also the culture-independent approaches. Discussion  It was evident that more representative fungal genera appeared in organic treatments than other treatments, indicating that culturable fungi were more sensitive to organic than to chemical fertilizers. A very notable finding was that fungal CFUs appeared maximal in organic manure treatments. This was quite different from the bacterial CFUs in the manure, indicating that bacteria and fungi responded differently to the fertilization. Similar to bacteria, the minimum fungal CFUs were also observed in the NK treatment. This result provided evidence that phosphorus could be a key factor for microorganisms in the soil. Thus, despite the fact that culture-dependent techniques are not ideal for studies of the composition of natural microbial communities when used alone, they provide one of the more useful means of understanding the growth habit, development, and potential function of microorganisms from soil habitats. A combination of culture-dependent and culture-independent approaches is likely to reveal more complete information regarding the composition of soil microbial communities. Conclusions  Long-term fertilization had great effects on the soil bacterial and fungal communities. Organic fertilizer applications induced the least culturable bacterial CFUs but the highest bacterial diversity, while chemical fertilizer applications had less impact on soil bacterial community. The largest fungal CFUs were obtained, but much lower diversity was detected in the manure treatment. The lowest bacterial and also fungal CFUs were observed in the NK treatment. The long-term fertilization treatments resulted in different bacterial and fungal compositions ascertained by the culture-dependent and also the culture-independent approaches. Phosphorus fertilizer could be considered as a key factor to control the microbial CFUs and diversity in this Chinese upland red soil. Recommendations and perspectives  Soil fungi seem to be a more sensitive indicator of soil fertility than soil bacteria. Since the major limitation of molecular methods in soil microbial studies is the lack of discrimination between the living and dead, or active and dormant microorganisms, both culture-dependent and culture-independent methods should be used to appropriately characterize soil microbial diversity.