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Keywords:

  • Denitrification;
  • Soil;
  • Fertilizer;
  • Diversity;
  • T-RFLP;
  • nir genes

Abstract

Temporal and spatial variation of communities of soil denitrifying bacteria at sites receiving mineral fertilizer (60 and 120 kgNha−1year−1) and cattle manure (75 and 150 kgNha−1year−1) were explored using terminal restriction fragment length polymorphism (T-RFLP) analyses of PCR amplified nitrite reductase (nirK and nirS) gene fragments. The analyses were done three times during the year: in March, July and October. nirK gene fragments could be amplified in all three months, whereas nirS gene fragments could be amplified only in March. Analysis of similarities in T-RFLP patterns revealed a significant seasonal shift in the community structure of nirK-containing bacteria. Also, sites treated with mineral fertilizer or cattle manure showed different communities of nirK-containing denitrifying bacteria, since the T-RFLP patterns of soils treated with these fertilizers were significantly different. Also, these sites significantly differed from the control plot (no fertilizer treatment), whereas the patterns for low and high N-additions were barely separable from each other. Sequencing and phylogenetic analysis of 54 nirK clones revealed that the major part of the nirK-containing bacteria investigated belonged to a yet uncultivated cluster of denitrifying bacteria.