Populations of methanogenic bacteria in paddy field soil under double cropping conditions (rice-wheat)

The methanogenic populations able to use H₂–CO₂, methanol, and acetate were investigated in paddy field soil in situ under double cropping conditions [rice (Oryza sativa L.) as a summer crop under flooded conditions and wheat (Triticum aestivum L.) as an upland winter crop] over 2 years approximately bimonthly by the most probable number method. Three fields, one without fertilizer, one treated with inorganic fertilizer (mixed fertilizer including urea, ammonium phosphate, and potassium sulfate), and one treated with wheat straw plus inorganic fertilizer, were examined. The population of H₂–CO₂, methanol, and acetate utilizers in the paddy field soil at a depth of 1–6 cm was 10³–10⁴, 10⁴–10⁵, and 10⁴–10⁵ g^-1 dry soil, respectively. These values were almost constant during the 2 years irrespective of moisture regime (flooded or nonflooded), crop (rice or wheat), fertilizer treatment, and soil depth (0–1, 1–10, and 10–20 cm).     

模拟干湿交替对水稻土古菌群落结构的影响

干湿交替是自然界普遍存在的现象,但长期以来由于技术的限制,复杂土壤中微生物对水分变化的响应规律仍不清楚。针对我国江苏常熟湖泊底泥发育的典型水稻土,在室内开展湿润-风干以及风干-湿润各三次循环,每次循环中湿润、风干状态各维持7d,利用微生物核糖体rRNA的通用引物进行PCR扩增,通过高通量测序分析土壤古菌多样性变化,同时结合实时荧光定量PCR技术,在DNA和RNA水平研究古菌数量对干湿交替过程的响应规律。结果表明:水稻土湿润-风干过程中,在DNA水平土壤古菌数量降幅约为149倍~468倍,而在RNA水平降幅最高仅为2.06倍;水稻土风干-湿润过程中,在DNA水平古菌数量增幅在147倍~360倍之间,而在RNA水平增幅最高仅为2.95倍。表明在干湿交替过程中,DNA水平的古菌16S rRNA基因数量变化远高于RNA水平。基于高通量测序多样性的结果表明,在DNA和RNA水平,湿润土壤3次风干、以及风干土壤3次加水湿润7d恢复后,土壤古菌群落结构均发生统计显著性改变。在微生物门、纲、目、科和属的不同分类水平下,水稻土古菌主要包括3、10、13、14、10种不同的类群,在RNA和DNA水平的结果基本一致。干湿交替导致部分古菌类群发生显著变化,其中在微生物分类学目水平发生显著变化的古菌最高达到6种,主要包括产甲烷古菌和氨氧化古菌,如Methanobacteriales、Methanosarcinales、Methanomicrobiales和Nitrososphaerales等。这些研究结果表明,反复的干湿交替并未显著改变水稻土中古菌的主要类群组成,古菌类群的绝对数量和相对丰度发生了一定程度的变化,但这些变化与微生物生理作用的联系仍需进一步研究;风干土壤中古菌RNA序列极可能来自于完整的古菌细胞,暗示了这些古菌细胞能够较好地适应水稻土中水分的剧烈变化,风干状态的土壤在一定程度也可用于土壤古菌群落组成研究。     

Community structure of microaerophilic iron-oxidizing bacteria in Japanese paddy field soils

ABSTRACT

Redox cycle of iron (Fe) is the central process in the biogeochemistry of paddy field soil. Although Fe(II)-oxidizing process is mediated by both abiotic and biotic reactions, microorganisms involved in the process have not been well studied in paddy field soil. The present study investigated the community structure of microaerophilic Fe(II)-oxidizing bacteria (FeOB) in the family Gallionellaceae in the plow layer of paddy fields located in the central (Anjo) and northeastern (Omagari) Japan since the members in the family are the typical FeOB in circumneutral freshwater environments and possibly have the significant role for Fe(II) oxidation in paddy field soils. A primer set targeting 16S rRNA gene of Gallionella-related FeOB was newly designed for the polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) and quantitative PCR (qPCR) analyses. DGGE analysis showed significant differences in the band patterns between the field sites. Besides, minor differences were observed in the patterns between the soil depths (0–1 cm and below 1 cm) in the Anjo field, while the patterns were relatively stable in the Omagari field during the annual rice cultivation practices. In total 54 bands were sequenced and clustered into 20 operational taxonomic units (OTUs) on the basis of the 97% similarity. Eighteen out of twenty OTUs (50 of 54 bands) were affiliated within the FeOB cluster of Gallionellaceae, some of which were clustered with known microaerophilic FeOB, Ferrigenium kumadai, Ferriphaselus amnicola, ‘Sideroxydans lithotrophicus’ and ‘S. paludicola’. The number of the 16S rRNA gene copies was 10⁵–10⁷ and 10⁶–10⁸ copies g^?1 dried soil in the two paddy fields and negatively correlated to the contents of acetate-extractable Fe(II) in the soils during the rice cultivation period. These results suggested inhabitance of considerable number of diverse Gallionella-related FeOB and their potential involvement in the Fe(II)-oxidizing process of soil, especially during the rice cultivation period in the paddy field soils.     

Effect of winter-flooding on methanogenic archaeal community structure in paddy field under organic farming

Paddy field is a major emission source of methane. Methane is the terminal product of anaerobic decomposition of organic matter and generated by methanogenic archaea under flooded conditions in paddy fields. This study aimed to reveal the effect of winter flooding on methanogenic archaeal community structure in paddy fields of Andosols under organic farming. Soil samples were collected from experimental paddy fields in the Field Science Center, Tohoku University, for two years. They were under flooding conditions during winter with organic farming, under non-flooding conditions during winter with organic farming and under non-flooding conditions during winter with conventional farming (non-organic farming). Polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) analysis of methanogenic archaeal 16S rRNA gene revealed that the DGGE patterns were nearly the same irrespective of the treatment and sampling times. Twenty-three bands were observed from each treatment and 4, 13 and 6 sequences were closely related to Methanomicrobiales, Methanosarcinales and Methanocellales, respectively. Real-time quantitative PCR analysis indicated that the abundance of methanogenic archaeal 16S rRNA gene and mcrA gene, encoding α subunit of methyl-coenzyme M reductase, was not significantly different among the paddy fields. This study first revealed a methanogenic archaeal community in an Andosol paddy field and showed that the community was not affected by winter flooding under organic farming.     

Phylogenetic study on a bacterial community in the floodwater of a Japanese paddy field estimated by sequencing 16S rDNA fragments after denaturing gradient gel electrophoresis

Phylogenetic positions of characteristic bands of 16S rDNA that were obtained from the floodwater of a Japanese paddy field by denaturing gradient gel electrophoresis (DGGE) analysis in a previous work (Biol Fertil Soils 36:306–312, 2002) were determined to identify dominant bacterial members in the floodwater. Sequences of DGGE bands were affiliated with the Cytophaga–Flavobacterium–Bacteroides group, β-Proteobacteria, and Actinobacteria and showed phylogenetically close relationships with species inhabiting other aquatic environments, although most of their closest relatives were uncultured bacterial clones.     

Eukaryotic communities associated with the decomposition of rice straw compost in a Japanese rice paddy field estimated by DGGE analysis

To estimate the succession and phylogenetic composition of the eukaryotic communities responsible for the decomposition of rice straw compost under flooded conditions during the cultivation period of paddy rice, denaturing gradient gel electrophoresis (DGGE) analysis targeting 18S rDNA followed by sequencing was conducted in a Japanese paddy field. The eukaryotic communities in rice straw compost incorporated into the flooded paddy field were influenced by the mid-season drainage and mainly composed of fungi (Ascomycota, Zygomycota, and Chytridiomycota) and protozoa (Ciliophora, Euglyphida, and Dactylopodida), most of which existed continuously during the cultivation period of paddy rice. The results indicated that these eukaryotic members were associated with the decomposition of rice straw compost in paddy field soil directly or indirectly.     

Application of DGGE analysis to the study of bacterial community structure in plant roots and in nonrhizosphere soil

To analyze the structure of bacterial communities in spinach roots and in the nonrhizosphere soil, we used PeR-amplified 16S rRNA gene fragments separated by denaturing gradient gel electrophoresis (DGGE). DGGE revealed a large number of band patterns, which were ascribed to various bacterial species composing each of the bacterial communities. The pattern from the roots was less complex than that from the soil. It is considered that DGGE analysis is suitable for studies of bacterial community structure in soil-plant ecosystems.     

Cyanobacterial communities of rice straw left on the soil surface of a paddy field

To estimate diversity, seasonal variation, and phylogeny of the cyanobacterial communities in rice straw placed in nylon mesh bags and left on the soil surface of a paddy field, total DNA was extracted from straw, amplified by polymerase chain reaction targeting 16S rRNA genes of cyanobacteria, and the amplicons were separated by denaturing gradient gel electrophoresis (DGGE). These DGGE bands were sequenced. The paddy field was under flooded condition after transplanting of rice (Experiment 1) and under drained conditions after harvest (Experiment 2). The residual samples on the soil surface under upland conditions were collected just before spring plowing and were placed again on the soil surface after transplanting under flooded conditions. DGGE band patterns of cyanobacterial communities of rice straw were different under drained conditions, under flooded conditions when fresh rice straw samples were placed (Experiment 1), and under flooded conditions when residual rice straw samples were replaced (Experiment 2), indicating that the communities were influenced by both water regime of the paddy field and the degree of the rice straw decomposition. Sequence analysis of DGGE bands indicated that most of the cyanobacteria in rice straw on the soil surface in the paddy field were filamentous members belonging to Subsections III and IV. Filamentous cyanobacterial cells were observed in rice straw under flooded conditions by epifluorescence microscopy.     

Effect of bensulfuron-methyl (a sulfonylurea herbicide) on the soil bacterial community of a paddy soil microcosm

The effect of bensulfuron-methyl (BSM) on a soil microbial community in a model paddy microcosm was studied. Total bacterial numbers in the overlying water and surface soil were monitored for 2 months after the application of BSM at the field rate and a ten-fold field rate. Pentachlorophenol (PCP) was used for comparison. Neither chemical affected the total bacterial numbers remarkably, either in the overlying water or in the surface soil. In contrast, the nitrification potential was significantly suppressed by the BSM application. The bacterial community structure, as evaluated by the denaturing gradient gel electrophoresis (DGGE) of PCR amplification products from bacterial 16S rDNA, was unaffected by the BSM treatments over 8 weeks in the surface soil, compared with the control (no pesticide). In contrast, the surface soil exposed to PCP at a ten-fold field rate showed different patterns from the controls at 4 weeks and 8 weeks after application. The DGGE patterns of the overlying water were much more variable than those of the surface soil in any treatments. Cluster analysis showed that the BSM plots were classified within the same group as the control at 1 week after application and that the BSM and PCP plots from 2 weeks onward after application were grouped differently from the control. Of 22 clones excised from the DGGE gels, 20 clones belonged to the Proteobacteria and two belonged to the Verrucomicrobia. It was considered that the impact of BSM on the overall microbial community (total numbers, community structure of soil) was negligible, although BSM had an impact on some specific functions of the soil microbial community (nitrification) and a part of the community (overlying water).     

Rapid changes in the rhizosphere bacterial community structure during re-colonization of sterilized soil

Diversity has been shown to be pivotal in ecosystem stability and resilience. It is therefore important to increase our knowledge about the development of diversity. The aim of this study was to investigate the temporal dynamics of the bacterial community structure in the rhizosphere of wheat plants growing in a soil in which the initial conditions for bacterial re-colonization were modified by mixing different amounts of sterilized with native soil at ratios of 19:1, 9:1, 4:1 and 1:1. Additional treatments comprised sterilized soil or native soil. Plant dry weight at day 20 decreased with increasing percentage of native soil in the mix. The bacterial community structure in the rhizosphere was assessed by polymerase chain reaction-denaturing gradient gel electrophoresis (DGGE) at days 3, 14 and 20 after planting. The bacterial community in the sterilized soil had a lower diversity and evenness than the native soil. Both diversity and evenness increased with time in the sterilized soil. Community structure in the different mixes changed over time and the changes were mix-specific. Principal component analyses of the DGGE banding patterns showed clear differences between the treatments particularly at day 3 and day 14 and revealed changes in community structure within a few days in a given treatment. The results of the present study show that bacterial communities rapidly re-colonize sterilized soil. During re-colonization, the community structure changes rapidly with a general trend towards higher diversity and evenness. The changes in community structure over time are also affected by the amount of sterile substrate to be re-colonized.     

Abundance and community composition of methanotrophs in a Chinese paddy soil under long-term fertilization practices

Background, aim, and scope  As the second most important greenhouse gas, methane (CH₄) is produced from many sources such as paddy fields. Methane-oxidizing bacteria (methanotrophs) consume CH₄ in paddy soil and, therefore, reduce CH₄ emission to the atmosphere. In order to estimate the contribution of paddy fields as a source of CH₄, it is important to monitor the effects of fertilizer applications on the shifts of soil methanotrophs, which are targets in strategies to combat global climate change. In this study, real-time polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE) based on 16S rRNA and pmoA genes, respectively, were used to analyze the soil methanotrophic abundance and community diversity under four fertilization treatments: urea (N), urea and potassium chloride (NK), urea, superphosphate, and potassium chloride (NPK), and urea, superphosphate, potassium chloride, and crop residues (NPK+C), compared to an untreated control (CON). The objective of this study was to examine whether soil methanotrophs responded to the long-term, different fertilizer regimes by using a combination of quantitative and qualitative molecular approaches. Materials and methods  Soil samples were collected from the Taoyuan Experimental Station of Agro-ecosystem Observation at Changde (28°55′ N, 111°26′ E), central Hunan Province of China, in July 2006. Soil DNAs were extracted from the samples, then the 16S rRNA genes were quantified by real-time PCR and the pmoA genes were amplified via general PCR followed by DGGE, cloning, sequencing, and phylogenetic analysis. The community diversity indices were assessed through the DGGE profile. Results  Except for NPK, other treatments of N, NK, and NPK+C showed significantly higher copy numbers of type I methanotrophs (7.0–9.6 × 10⁷) than CON (5.1 × 10⁷). The copy numbers of type II methanotrophs were significantly higher in NPK+C (2.8 × 10⁸) and NK (2.5 × 10⁸) treatments than in CON (1.4 × 10⁸). Moreover, the ratio of type II to type I methanotrophic copy numbers ranged from 1.88 to 3.32, indicating that the type II methanotrophs dominated in all treatments. Cluster analyses based on the DGGE profile showed that the methanotrophic community in NPK+C might respond more sensitively to the environmental variation. Phylogenetic analysis showed that 81% of the obtained pmoA sequences were classified as type I methanotrophs. Furthermore, the type I-affiliated sequences were related to Methylobacter, Methylomicrobium, Methylomonas, and some uncultured methanotrophic clones, and those type II-like sequences were affiliated with Methylocystis and Methylosinus genera. Discussion  There was an inhibitory effect on the methanotrophic abundance in the N and a stimulating effect in the NK and NPK+C treatments, respectively. During the rice-growing season, the type II methanotrophs might be more profited from such a coexistence of low O₂ and high CH₄ concentration environment than the type I methanotrophs. However, type I methanotrophs seemed to be more frequently detected. The relatively complex diversity pattern in the NPK+C treatment might result from the strong CH₄ production. Conclusions  Long-term fertilization regimes can both affect the abundance and the composition of the type I and type II methanotrophs. The inhibited effects on methanotrophic abundance were found in the N treatment, compared to the stimulated effects from the NK and NPK+C treatments. The fertilizers of nitrogen, potassium, and the crop residues could be important factors controlling the abundance and community composition of the methanotrophs in the paddy soil. Recommendations and perspectives  Methanotrophs are a fascinating group of microorganisms playing an important role in the biogeochemical carbon cycle and in the control of global climate change. However, it is still a challenge for the cultivation of the methanotrophs, although three isolates were obtained in the extreme environments very recently. Therefore, future studies will be undoubtedly conducted via molecular techniques just like the applications in this study.     

Community structure of bacteria on different types of mineral particles in a sandy soil

Various types of mineral particles in a soil probably provide different microenvironments for microorganisms. The purpose of this study is to investigate whether different types of mineral in a soil harbor different bacterial populations. DNA was extracted from five types (quartz, feldspar, pyroxene, magnetite, iron-coated reddish brown particles) of sand-size mineral particles separated from a sandy soil, and was amplified for partial 16 S rRNA gene by polymerase chain reaction (PCR). Twenty-nine to 69 amplicons per each type of mineral were cloned and sequenced, followed by phylogenetic affiliation of the sequences. As a result, some types of bacteria were detected on all of the types of mineral including the orders Rhizobiales, Bacillales, and Acidobacteriales. In the case of Acidobacteriales, higher percentages were found on magnetite and quartz. Some taxa were restricted to specific types of mineral; the class Actinobacteria was found on pyroxene but not on quartz, and rarely on magnetite and feldspar. Bacterial diversity at the order level estimated by Chao1 value was higher in feldspar and pyroxene than the other three types of mineral. The UniFrac Significance test indicated that the differences in bacterial communitiy structures among the particles were suggestive except that between feldspar and pyroxene. These results support the idea that different communities of bacteria were associated with each of the mineral types.     

Long-term fertilization regimes affect bacterial community structure and diversity of an agricultural soil in northern China

Background, Aims, and Scope  Knowledge about shifts of microbial community structure and diversity following different agricultural management practices could improve our understanding of soil processes and thus help us to develop sound management strategies. A long-term fertilization experiment was established in 1989 at Fengqiu (35°00′N, 114°24′E) in northern China. The soil (sandy loam) is classified as aquic inceptisols and has received continuous fertilization treatments since then. The fertilization treatments included control (CK, no fertilizer), chemical fertilizers nitrogen (N) and potassium (K) (NK), phosphorous (P) and K (PK), NP, NPK, organic manure (OM), and half chemical fertilizers NPK plus half organic manure (1/2NPKOM). The objective of this study was to examine if the microbial community structure and diversity were affected by the long-term fertilization regimes. Materials and Methods  Soil samples were collected from the long-term experimental plots with seven treatments and four replications in April 2006. Microbial DNAs were extracted from the soil samples and the 16S rRNA genes were PCR amplified. The PCR products were analyzed by DGGE, cloning and sequencing. The bacterial community structures and diversity were assessed using the DGGE profiles and the clone libraries constructed from the excised DGGE bands. Results  The bacterial community structure of the OM and PK treatments were significantly different from those of all other treatments. The bacterial community structures of the four Ncontaining treatments (NK, NP, NPK and 1/2NPKOM), as well as CK, were more similar to each other. The changes in bacterial community structures of the OM and PK treatments showed higher richness and diversity. Phylogenetic analyses indicated that Proteobacteria (30.5%) was the dominant taxonomic group of the soil, followed by Acidobacteria (15.3%), Gemmatimonadetes (12.7%), etc. Discussion  Irrespective of the two fertilization treatments of OM and PK, the cluster analysis showed that bacterial communities of the remaining five treatments of CK, NK, NP, NPK and 1/2NPKOM seemed to be more similar to each other, which indicated the relatively weak effects of the four N-containing treatments on soil bacterial communities. N fertilizer may be considered as a key factor to counteract the effects of other fertilizers on microbial communities. Conclusions  Our results show that long-term fertilization regimes can affect bacterial community structure and diversity of the agricultural soil. The OM and PK treatments showed a trend towards distinct community structures, higher richness and diversity when compared to the other treatments. Contrasting to the positive effects of OM and PK treatments on the bacterial communities, N fertilizer could be considered as a key factor in the soil to counteract the effects of other fertilizers on soil microbial communities. Recommendations and Perspectives  Because of the extremely high abundance and diversity of microorganisms in soil and the high heterogeneity of the soil, it is necessary to further examine the effects of fertilization regimes on microbial community and diversity in different type soils for comprehensively understanding their effects through the appropriate combination of molecular approaches. ESS-Submission Editor: Chengrong Chen, PhD (c.chen@griffith.edu.au)     

Overland flow systems for treatment of landfill leachates—Potential nitrification and structure of the ammonia-oxidising bacterial community during a growing season

Overland flow systems are useful for treating landfill leachates, because they provide favourable conditions for nitrification and they are easy to maintain. However, little is known about the microbial communities in such systems or the nitrification capacity of those microorganisms. In this study, seasonal variations in potential nitrification and in community composition of nitrifying bacteria were investigated in two overland flow areas receiving leachate from landfills at Korslöt and Hagby, Sweden. Samples were collected in the settling ponds sediment and at two depths in the overland flow areas (the macrophyte litter layer and the rhizosphere) in May, August and November 2003. A short-term incubation method was used to measure potential oxidation of ammonia and nitrite (designated PAO and PNO). The ammonia-oxidising bacterial (AOB) community was investigated using a 16S rRNA gene approach that included PCR amplification and analysis of PCR products by denaturing gradient gel electrophoresis (DGGE), followed by nucleotide sequencing and phylogenetic analysis.PAO was determined in the range 5-2700 (NO₂⁻+NO₃⁻)-N g⁻¹ dw d⁻¹ and PNO in the range 60-2000 μg NO₂⁻-N g⁻¹ dw d⁻¹. At Korslöt, PAO and PNO showed similar temporal variation in the different ecosystems, whereas no such relationship was noticed at Hagby. Considering both sites, there was no obvious change in the composition of the AOB community over the growing season. However, the composition did differ between the ecosystems: Nitrosomonas-like sequences were more common in the ponds, and in the litter layers they were found as often as Nitrosospira-like sequences, whereas Nitrosospira-like sequences were more common in the rhizospheres. Altogether, we found nine different AOB sequences, five Nitrosomonas-like and four Nitrosospira-like, which belonged to clusters 0, 2, 3b, 6a, 6b and 7. There was no apparent relationship between the number of AOB populations and the PAO in different soil layers and sediments.     

抗真菌转基因水稻根际土壤真菌群落结构的动态变化

以非转基因水稻"七丝软粘"为对照,采用传统平板计数法和变性梯度凝胶电泳技术,研究了抗真菌转基因水稻"转品1"和"转品8"生长周期内对根际土壤中可培养真菌数和真菌群落结构的影响。结果显示,相同生育期转基因水稻根际土壤可培养真菌数量与其非转基因对照水稻相比较无显著性差异,表明转基因水稻的种植没有对根际土壤真菌数量产生明显影响;18S rRNA真菌群落DGGE图谱分析显示,相同生育期转基因水稻与其非转基因对照水稻的根际土壤真菌DGGE条带数量和条带位置均无显著性差异,表明转基因水稻的种植没有对根际土壤真菌群落结构产生明显影响。进一步分析相同生育期转基因水稻与其非转基因对照水稻的根际土壤真菌群落香农多样性指数(Shannon diversity index)和均匀度指数(Evenness index)的动态变化,发现两者均没有显著性差异。以上研究结果表明,外源抗真菌基因的导入对水稻根际土壤中真菌群落数量和群落结构均没有明显影响。此外,将不同位置的真菌DGGE条带切胶回收,克隆、测序后,进行系统进化树分析,结果表明根际土壤真菌群落主要归属为子囊菌门(Ascomycota)、担子菌门(Basidiomycota)、壶菌门(Chytridiomycota)、接合菌门(Zygomycota)和未知真菌(unknown fungi)5个类群。     

Design and evaluation of nematode 18S rDNA primers for PCR and denaturing gradient gel electrophoresis (DGGE) of soil community DNA

Consensus nematode 18S ribosomal DNA primers were designed by aligning available 18S sequences and identifying a variable region flanked by highly conserved regions. These primers were then used to amplify nematode 18S rDNA from whole soil community DNA extracted from a range of European grassland types. Cloning of the PCR amplicons (778 bp) followed by restriction digest analysis (RFLP) resulted in the recovery of 34 unique nematode sequences from the four grasslands studied. Comparison of these data with the limited number of 18S rDNA nematode sequences currently held in on-line databases revealed that all of the sequences could be assigned to known nematode taxa albeit tentatively in some cases. Two of the sequences recovered from the site in the Netherlands (wet, hay-grassland) were recovered in a clade that included a sequence of the genus Trichodorus whilst other sequences from this site showed similarity with 18S rDNA sequences of the genus Prismatolaimus (five sequences), Xiphinema (one sequence) and Enoplus (one sequence). Of the remaining sequences, two showed some affinity with Mylonchulus (UK, upland peat), four with Steinernema (UK) and one sequence with Mesorhabditis (Hungary, east European Steppe). Three sequences from the Netherlands and one from Hungary were recovered in a clade that included a sequence of the genus Pratylenchoides whilst three further sequences from the Netherlands and two from Hungary were recovered in a clade encompassing the genus Globodera. Of the remaining nine sequences, two (NL6, NL62) formed a distinct lineage within the Adenophorea with 90% bootstrap recovery in a paraphyletic clade that included sequences of Prismatolaimus and Trichodorus. Seven sequences (three from the Netherlands, three from the UK and one from Greece) were left unassigned though the tree topology suggested some relationship (58% bootstrap recovery) with the genus Cephalobus. To assess whether primers used to amplify 18S rDNA might be used to fingerprint genetic diversity in nematode communities in soil, the environmental sequence data were used to design a second set of primers carrying a GC-clamp. These primers amplified a 469 bp fragment internal to the region flanked by the primer set used to derive the nematode trees and were used to amplify 18S rDNA for subsequent analysis using denaturing gradient gel electrophoresis (DGGE). DGGE analysis of six major European grassland types revealed considerable genetic diversity between sites. However, the relationships seen with the DGGE data were inconsistent with previous studies where the same soils had been characterized with respect to functional and morphological diversity. To confirm that this second set of primers was amplifying nematode sequences, selected bands on the DGGE gels were extracted, PCR amplified and sequenced. The final alignment was 337 bases. These analyses revealed the presence of sequence signatures from the genera Paratrichodorus, Plectus, Steinernema, Globodera, Cephalobus and Pratylenchoides.