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.

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) MPNs 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.

Two hypotheses on repression of nitrification in climax vegetations (i.e. nitrogen immobilization and allelopathy) were investigated. In this study the potential nitrification activities and numbers of ammonium‐oxidizing bacteria were established in a nature reserve with a series of natural grasslands with vegetational different stages of succession of plants species. The pastures had not been fertilized for 3, 7, 20 and 46 years, respectively, and the gradual decrease in availability of nutrients had led to pastures dominated by different grass species. In each field soil parameters, potential nitrification activities (PNA) and numbers of ammonium‐oxidizing bacteria were determined in the root zone of Holcus lanatus as well as in that of a grass species characteristic of the stage of succession. In the rhizosphere of H. lanatus decreasing PNA and numbers of ammonium‐oxidizing bacteria were observed as the period of non fertilization increased. Within each field no significant differences in PNA were observed between the root zones of H. lanatus and those of the dominant grass species. From these results it is concluded that, in these fields, decreasing nitrification was related only to decreasing ammonium availability and not to species composition. No indications were obtained that allelochemicals were involved in the flow nitrification potentials of late stages of succession. The optimum pH of the ammonium‐oxidizing community, measured as PNA, decreased as the period of non fertilization increased. It is suggested that impoverishment of the grassland soil with respect to nitrogen availability selects against ammonium‐oxidizing bacteria with a relatively high pH optimum.

Two hypotheses on repression of nitrification in climax vegetations (i.e. nitrogen immobilization and allelopathy) were investigated. In this study the potential nitrification activities and numbers of ammonium‐oxidizing bacteria were established in a nature reserve with a series of natural grasslands with vegetational different stages of succession of plants species. The pastures had not been fertilized for 3, 7, 20 and 46 years, respectively, and the gradual decrease in availability of nutrients had led to pastures dominated by different grass species. In each field soil parameters, potential nitrification activities (PNA) and numbers of ammonium‐oxidizing bacteria were determined in the root zone of Holcus lanatus as well as in that of a grass species characteristic of the stage of succession. In the rhizosphere of H. lanatus decreasing PNA and numbers of ammonium‐oxidizing bacteria were observed as the period of non fertilization increased. Within each field no significant differences in PNA were observed between the root zones of H. lanatus and those of the dominant grass species. From these results it is concluded that, in these fields, decreasing nitrification was related only to decreasing ammonium availability and not to species composition. No indications were obtained that allelochemicals were involved in the flow nitrification potentials of late stages of succession. The optimum pH of the ammonium‐oxidizing community, measured as PNA, decreased as the period of non fertilization increased. It is suggested that impoverishment of the grassland soil with respect to nitrogen availability selects against ammonium‐oxidizing bacteria with a relatively high pH optimum.

The results of Most Probable Number determinations applying low and high concentrations of nitrite reveal the presence of at least two different communities of potential nitrite‐oxidizing bacteria in a number of soil types. The effect of plant roots on these two communities was studied in pot experiments with soil from natural grassland in the presence or absence of either Festuca rubra or Plantago lanceolata. Both plant species are dominant on the grassland soil used in this study. Plant roots had a stimulating effect on the numbers of nitrite‐oxidizing bacteria determined with 0.05 mM nitrite in the enumeration medium as well as on the potential nitrite‐oxidizing activity. On the other hand, plants roots, especially in younger plants, repressed the numbers of nitrite‐oxidizing bacteria enumerated with 5.0 mM nitrite in the counting medium. Pure culture studies with organotrophically grown Nitrobacter species clearly showed that this type of potential nitrite‐oxidizing bacteria could not have been responsible for the relatively high Most Probable Numbers observed in the root zones when applying 0.05 mM nitrite in the enumeration medium.

The results of Most Probable Number determinations applying low and high concentrations of mtrlte reveal the presence of at least two different communities of potential mtnte-oxldlZlng bacteria in a number of soil types The effect of plant roots on these two communities was studied in pot experiments with soil from natural grassland in the presence or absence of either Festuca rubra or Plantago lanceolata. Both plant species are dominant on the grassland soil used in this study Plant roots had a stimulating effect on the numbers of nitrite-oxidizing bacteria determined with 0 05 mM nitrite m the enumeration medium as well as on the potential nitrite-oxidizing activity. On the other hand, plants roots, especially in younger plants, repressed the numbers of nitrite-oxidizing bacteria enumerated with 5 0 mM nitrite in the counting medium Pure culture studies with organotrophlcally grown Nttrobacter species clearly showed that this type of potential nitrite-oxidizing bacteria could not have been responsible for the relatively high Most Probable Numbers observed in the root zones when applying 0 05 mM nitrite m the enumeration medium