Search

Changes in the community structure of chemolitho-autotrophic ammonia-oxidising bacteria of the beta-subgroup Proteobacteria were monitored during nutrient-impoverishment management of slightly acidic, peaty grassland soils, which decreased in pH with succession. Specific PCR, cloning and sequence analysis. denaturing gradient gel electrophoresis (DGGE) and probe hybridisation were used to analyse rDNA sequences directly recovered from successional soils. Four previously characterised ammonia oxidiser sequence clusters were recovered from each soil, three associated with the genus Nitrosospira and one with the genus Nitrosomonas. All samples were dominated by Nitrosospira-like sequences, Nitrosospira cluster 3 was the most commonly recovered ammonia oxidiser group in all fields, but a greater representation of Nitrosospira clusters 2 and 4 was observed in older fields. Most probable number (MPN) counts were conducted using neutral and slightly acid conditions. Neutral pH (7.5) MPN's suggested a decrease in ammonia oxidiser numbers in later successional fields, but this trend was not observed using slightly acid (pH 5.8) conditions. Analysis of terminal MPN dilutions revealed a distribution of sequence clusters similar to direct soil DNA extractions. However, an increased relative recovery of Nitrosospira cluster 2 was observed for acid pH MPNs compared to neutral pH MPNs from the most acidic soil tested, in agreement with current hypotheses on the relative acid tolerance of this group. [KEYWORDS: beta-subgroup proteobacteria; soil pH; nitrification; DGGE; most probable number (MPN); oligonucleotide hybridisation; Nitrosospira; Nitrosomonas 16s ribosomal-rna; gradient gel-electrophoresis; oxidizing bacteria; nitrosomonas-europaea; gene-sequences; populations; ph; nitrification; diversity; pcr]

A study was carried out to determine whether expansion of marram-grass stands (Ammophila arenaria (L.) Link) on acidic inner Dutch coastal dunes was caused by suppressiveness of soils from these stands against three potential pathogenic fungi of marram grass, namely Fusarium culmorum (W. G. Sm.) Sacc., Phoma exigua Desm. and a Ulocladium sp. The suppressiveness of the acidic inner dune soils was compared with that of lime-rich dune soils from vigorous and declining marram-grass stands. Suppressiveness of the dune soils against the saprotrophic fungi Chaetomium globosum Kunze: Fr, Mucor hiemalis Wehmer and Trichoderma harzianum Rifai was also determined. All fungi had been isolated from marram-grass roots. Suppressiveness was determined by comparing the formation of hyphae from potato-dextrose agar discs into (layer method) or on top of (surface method) dune soils with that of controls consisting of sterile, acid-washed beach sand. The growth of the three root-infecting fungi was strongly inhibited in all soils regardless of the method used. Hence, there were no indications that the potential pathogenic fungi were selectively suppressed by the acidic dune soils and, consequently, the results did not give any indication for the involvement of a fungal component in the decline of marram grass. Growth of the saprotrophs C. globosum and M. hiemalis was much less inhibited than that of the root-infecting fungi. Growth of T. harzianum was strongly inhibited in alkaline soils but not in the acid ones. The suppression of fungal growth could be partly or completely eliminated by a microwave treatment, indicating that biological components of the soil were essential to suppressiveness. The suppression of the fungi by colonies of dune soil micro-organisms on water-agar differed considerably from soil alone. Yet, all methods indicated the occurrence of general suppressiveness against fungi by dune soils, irrespective of the origin of the soil samples. This suppressiveness was probably not due to direct competition with other soil micro-organisms for nutrients but to inhibiting compounds produced by the soil micro-organisms. [KEYWORDS: suppressiveness; antagonism; dune soils; Ammophila arenaria (L.) Link Plant-parasitic nematodes; coastal foredunes; l link; vegetation; degeneration; grassland; heathland; organisms;bacteria]

The community structure of beta-subclass Proteobacteria ammonia-oxidizing bacteria was determined in semi-natural chalk grassland soils at different stages of secondary succession. Both culture-mediated (most probable number; MPN) and direct nucleic acid-based approaches targeting genes encoding 16S rRNA and the AmoA subunit of ammonia monooxygenase were used. Similar shifts were detected in the composition of the ammonia oxidizer communities by both culture-dependent and independent approaches. A predominance of Nitrosospira sequence cluster 3 in early successional fields was replaced by Nitrosospira sequence cluster 4 in late successional fields. The rate of this shift differed between the two areas examined. This shift occurred in a background of relative stability in the dominant bacterial populations in the soil, as determined by domain- level polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE). Molecular analysis of enrichment cultures obtained using different ammonia concentrations revealed biases towards Nitrosospira sequence cluster 3 or Nitrosospira sequence cluster 4 under high- or low-ammonia conditions respectively. High-ammonia MPNs suggested a decease in ammonia oxidizer numbers with succession, but low-ammonia MPNs and competitive PCR targeting amoA failed to support such a trend. Ammonia turnover rate, not specific changes in plant diversity and species composition, is implicated as the major determinant of ammonia oxidizer community structure in successional chalk grassland soils. [KEYWORDS: 16s ribosomal-rna; gradient gel-electrophoresis; nitrosomonas-europaea; nitrifying bacteria; species composition; gene-sequences; dna fragments; populations; nitrification; soil]

Denaturing gradient gel electrophoresis (DGGE) is a powerful and convenient tool for analyzing the sequence diversity of complex natural microbial populations, DGGE was evaluated for the identification of ammonia oxidizers of the beta subdivision of the Proteobacteria based on the mobility of PCR-amplified 16S rDNA fragments and for the analysis of mixtures of PCR products from this group generated by selective PCR of DNA extracted from coastal sand dunes, Degenerate PCR primers, CTO189f-GC and CTO654r, incorporating a 5' GC clamp, were designed to amplify a 465-bp 16S rDNA region spanning the V-2 and V-3 variable domains, The primers were tested against a representative selection of clones and cultures encompassing the currently recognized P-subdivision ammonia oxidizer 16S rDNA sequence diversity, Analysis of these products by DGGE revealed that while many of the sequences could be separated, some which were known to be different migrated similarly in the denaturant system used, The CTO primer pair was used to amplify 16S rDNA sequences from DNA extracted from soil sampled from Dutch coastal dune locations differing in pH and distance from the beach, The derived DGGE patterns were reproducible across multiple DNA isolations and PCRs, Ammonia oxidizer-like sequences from different phylogenetic groupings isolated from gene libraries made from the same sand dune DNA samples but prepared with different primers gave DGGE bands which comigrated with most of the bands detected from the sand dune samples, Bands from the DGGE gels of environmental samples were excised, reamplified, and directly sequenced, revealing strong similarity or identity of the recovered products to the corresponding regions of library clones, Six of the seven recognized sequence clusters of beta-subdivision ammonia oxidizers were detected in the dune systems, and differences in community structure between some sample sites were demonstrated, The most seaward dune site contained sequences showing affinity with sequence clusters previously isolated only from marine environments and was the only site where sequences related to the Nitrosomonas genus could be detected. Nitrosospira-like sequences were present in all sites, and there was some evidence of differences between Nitrosospira populations in acid and alkaline dune soils, Such differences in community structure may reflect physiological differences within beta-subdivision ammonia oxidizers, with consequent effects on nitrification rates in response to key environmental factors. [KEYWORDS: Purple bacteria; rna; phylogeny; genes; samples; soils]

Three aspects of the nitrate production in natural ecosystems are discussed,i.e. the population biology of nitrifying bacteria, the nitrate-producing activity of these organisms and the uptake of nitrate by higher plants. It is concluded that the three methods used in enumerating the nitrifying bacteria,i.e. the Most Probable Number method, the Fluorescent Antibody technique and the Potential Nitrification Rate, all have serious drawbacks and count different segments of the nitrifying populations. From the number of nitrifying bacteria no reliable estimate of the rate production can be obtained and also estimates that are made using field-incubation and15N−NH 4 + techniques do not yield reliable data. Possibly the best results can be obtained using Schimel's method to estimate the actual nitrification rate using15N−NO 3 − , but this method has still not been tested under different sets of soil conditions. From the nitrate reductase activity and the chemical composition of the plant a picture can be obtained of the quantities of nitrate and ammonium that have been taken up. However, it is shown that nitrate and ammonium are taken up in different proportions that they are produced. It is concluded that the various parameters have to be studied simultaneously, preferably in defined systems with plants, in which the participating organisms are known.

Key words : Heterodera , plant-parasitic nematodes, soil pathogens, Ammophila arenaria , occurrence, abundance, specificity, population dynamics, life history, pathogenicity, PCR-SSCP, molecular method, escape, sand burial, dispersal, migration, fitness, development time, survival, reproductive success, bottom-up, top-down.In natural ecosystems hardly any attention has been given to the population dynamics of plant-parasitic nematodes. In coastal foredunes, plant-parasitic nematodes are supposed to be involved in the degeneration and succession of the dominant sand-fixing grass Ammophila arenaria (Marram grass). The specificity, pathogenicity and population dynamics of the sedentary endoparasitic nematode Heterodera arenaria have been studied to determine if this species might be a key component of the soil pathogen complex of A. arenaria.H. arenaria was found to be specific to Elymus farctus and A. arenaria in the mobile area of the coastal foredunes. Colonisation of the newly deposited sand layer by H. arenaria corresponded well with the development of pathogenicity in a series of bioassays. However, direct addition of the nematode to A. arenaria did not result in growth reduction of the plant. So, H. arenaria behaves like a biotrophic parasite, which has a high specificity but is not aggressive. Therefore, H. arenaria did not seem to be directly involved in the degeneration of A. arenaria .Each year, the majority of the population of new H. arenaria cysts develops in the newly deposited sand layers. These layers are colonised by A. arenaria roots throughout the growing season. Migration to the new root layer may offer an individual nematode the benefit of early development and a larger potential offspring. The continuous release of juveniles in the field and their development in experiments indicate that release of juveniles from cysts is an ultimately determined process. Juveniles were found to emerge in November and many eggs or juveniles did not survive the winter period. The strategy of release, however, seems effective; the distance of migration could be too large to detect specific cues from the plant and the start of root formation in the field is highly variable. The emergence of juveniles late in the growing season could result in a second generation within the same year. The constant number of cysts per gram of roots suggests that the population density of H. arenaria is most likely a bottom-up directed process.

Ammophila arenaria (Marram grass) is the most dominant sand-fixing plant species in the Dutch coastal foredunes. This species has a natural ability to emerge from being buried and is therefore used to stabilize the coastal foredunes. On seaward slopes where plants are buried regularly with windblown sand, plants retain their vigour, but start to degenerate when sand accumulation diminishes. One of the factors that may cause degeneration at stabilized sites is the infection of roots by nematodes and fungi. Burial by fresh windblown sand may enable the plants to overcome these harmful soil organisms. In the present study, the nature of the soil-borne disease and its relationship with sand deposition is investigated. In a field survey, a wide range of nematodes and fungi were isolated from the root zone of A. arenaria . Subsequent inoculation-experiments showed that adding single fungal species did not reduce the growth of seedlings whereas combining all commonly found fungi together did, thus indicating synergistic effects. Adding 80 times more individuals of the semi- endoparasitic nematode Telotylenchus ventralis than present in natural soil reduced the growth of seedlings to the same extent as in natural soil. Several groups of soil organisms, especially those groups that include plant-parasitic nematodes, have shown to affect the growth of A. arenaria Burial with unsterilized root zone sand was less beneficial for plant growth than burial with sterilized or beach sand. This implies that plants are able to escape infection by soil organisms through upward growth following sand accumulation. Fungi colonized the freshly deposited layer of sand faster than plant-parasitic nematodes. Furthermore, it could be shown that in windblown soil numbers of fungal propagules and nematodes were reduced. Rejuvenation of stands along the accumulating edges of blowouts can, therefore, be explained by the reduced inoculum pressure of plant-pathogenic organisms in the deposited soil. The amount of sand and the time when sand is deposited are important components in the chances of A. arenaria to escape infection by soil organisms.

Decline and succession in dune scrub vegetation of sea buckthorn, Hippophaë rhamnoides L. in the Netherlands was previously found to be stimulated by biotic soil factors. In the present study, the role of plant-parasitic nematodes and root fungi was investigated. A field survey showed that during the ageing of H. rhamnoides vegetation there was a change in the plant-feeding nematode fauna. Certain nematode species, such as Tylenchorhynchus microphasmis Loof, were present in different stages and their population increased from stage to stage when expressed per unit of root length. The fraction of root length infected by VA-mycorrhizal fungi increased, as did soil concentrations of nitrogen and organic matter. Pot experiments with selective inhibition of groups of soil organisms indicated that soil fungi could be harmful to H . rhamnoides in the presence of nematodes, although fungi were not harmful on their own. The impact of various densities of T . microphasmis, either alone or in combination with the nitrogen-fixing actinorhizal symbiont Frankia or the fungus Cylindrocarpon destructans Zinnsm. Scholten, was assessed in pot experiments with controlled soil moisture conditions. Results showed inhibitory effects of the nematode on growth and on the acquisition of certain nutrients (especially phosphate). Although root nodulation and nitrogen fixation by Frankia were reduced, this did not appear to be the primary cause of growth reduction. No synergic interaction was found between T . microphasmis and C. destructans in their effect on growth of H . rhamnoides. The results of this study support the hypothesis that the increase in the concentration of soil nitrogen in the field stimulates nematode activity which in its turn impedes the acquisition of phosphate by H. rhamnoides. This reduces the competitive ability of this shrub and gives way to other plant species in the vegetation which are less sensitive to root damage.