Impact of plant functional group, plant species, and sampling time on the composition of nirK-type denitrifier communities in soil

Abstract

We studied the influence of eight nonleguminous grassland plant species belonging to two functional groups (grasses and forbs) on the composition of soil denitrifier communities in experimental microcosms over two consecutive years. Denitrifier community composition was analyzed by terminal restriction fragment length polymorphism (T-RFLP) of PCR-amplified nirK gene fragments coding for the copper-containing nitrite reductase. The impact of experimental factors (plant functional group, plant species, sampling time, and interactions between them) on the structure of soil denitrifier communities (i.e., T-RFLP patterns) was analyzed by canonical correspondence analysis. While the functional group of a plant did not affect nirK-type denitrifier communities, plant species identity did influence their composition. This effect changed with sampling time, indicating community changes due to seasonal conditions and a development of the plants in the microcosms. Differences in total soil nitrogen and carbon, soil pH, and root biomass were observed at the end of the experiment. However, statistical analysis revealed that the plants affected the nirK-type denitrifier community composition directly, e.g., through root exudates. Assignment of abundant T-RFs to cloned nirK sequences from the soil and subsequent phylogenetic analysis indicated a dominance of yet-unknown nirK genotypes and of genes related to nirK from denitrifiers of the order Rhizobiales. In conclusion, individual species of nonleguminous plants directly influenced the composition of denitrifier communities in soil, but environmental conditions had additional significant effects.

FIG. 1.

Average relative abundances of nirK T-RFs from soil of replicate plant microcosms in summer 2003 (for data on individual microcosms, see Fig. S1 and S2 in the supplemental material). Peak size is given in base pairs, and the relative abundance of T-RFs is given as percentage of total peak height. Fragment sizes within the graph indicate the sizes (bp) of theoretical T-RFs of clones after in silico analysis. A. p., Alopecurus pratensis; A. e., Arrhenatherum elatius; H. l., Holcus lanatus; A. o., Anthoxanthum odoratum; P. l., Plantago lanceolata; T. o., Taraxacum officinale; R. a., Ranunculus acris; G. p., Geranium pratense.

FIG. 2.

CCA ordination plot for the effect of plant species identity on the composition of the nirK-type denitrifier community in soil, based on the relative abundances of nirK T-RFs from the soil of three replicate plant microcosms. Circles indicate plant species and triangles the T-RFs, which are labeled according to fragment size (bp). The eigenvalues of the first and second axes in the ordination diagram are as follows: λ₁ = 0.094; λ₂ = 0.029. A. p., Alopecurus pratensis; A. o., Anthoxanthum odoratum; A. e., Arrhenatherum elatius; H. l., Holcus lanatus; G. p., Geranium pratense; P. l., Plantago lanceolata; R. a., Ranunculus acris; T. o., Taraxacum officinale.

FIG. 3.

CCA ordination plot for the effect of sampling time (circles) on the composition of the nirK-type denitrifier community in soil. T-RFs (triangles, labeled according to fragment size [bp]) are based on nirK T-RFLP data from microcosms. The eigenvalues of the first and second axes in the ordination diagram are as follows: λ₁ = 0.076; λ₂ = 0.023.

FIG. 4.

Maximum-likelihood tree based on partial nirK sequences (432 nucleotide positions) from cultured nirK-type denitrifiers and sequences from microcosm soil. The tree topology is supported by parsimony and neighbor-joining methods. Dashed lines indicate multifurcations where the tree topology was not consistently resolved. Hl, clones obtained from soil of Holcus lanatus; Pl, clones obtained from soil of Plantago lanceolata. S3, summer 2003; H3, autumn 2003. Numbers after the name of the clone indicate the respective sizes of T-RFs after in silico analysis with the restriction enzyme HaeIII. Accession numbers are in parentheses.