Analysis of ammonia-oxidizing bacteria associated with the roots of Proteaceae plant species in soils of Fynbos ecosystem

Abstract

Molecular methods were used to investigate the microbial diversity and community structure of ammonia-oxidizing bacteria (AOB) associated with the roots of the Proteaceae plant family. The identification of ammonia oxidizing bacteria in this ecosystem is of particular interest since Proteaceae are adapted to acidic, low nutrient (e.g. nitrogen) soils. The ammonia monooxygenase operon was used as a molecular marker to identify ammonia-oxidizing bacteria associated with the proteoid roots of the three Proteaceae members and compared to non-plant associated soil. PCR amplification using primer sets targeting the ammonia monooxygenase gene (amoA subunits) were used to construct a clone library. Sequence diversity was determined by RFLP analysis of amoA to identify major groups of AOB of the ~-subclass of Proteobacteria in total community DNA, and DNA sequencing and phylogenetic analysis were also applied. DGGE analysis was performed to determine the community structure and distribution of ammonia-oxidizing bacteria in plant-associated and non-plant associated soils. The AOB genotypic diversity was similar in the plant-associated samples and non-plant associated soil. All AOB phylotypes belonged to Nitrosospira species and clustered with Nitrosospira cluster 3. The abundance of the amoA was quantified to be approximately 4.2 x 107 copies/g of dry soil, using a real-time PCR assay. These data suggest that the Nitrosospira species are the dominant phylotypes in that environment. This investigation provides new insights into the relationships between plants and ammonia-oxidizing bacteria in natural Fynbos ecosystems.