Analysis of nifH Gene Pool Complexity in Soil and Litter at a Douglas Fir Forest Site in the Oregon Cascade Mountain Range

Abstract

Nitrogen-fixing microbial populations in a Douglas fir forest on the western slope of the Oregon Cascade Mountain Range were analyzed. The complexity of the nifH gene pool (nifH is the marker gene which encodes nitrogenase reductase) was assessed by performing nested PCR with bulk DNA extracted from plant litter and soil. The restriction fragment length polymorphisms (RFLPs) of PCR products obtained from litter were reproducibly different than the RFLPs of PCR products obtained from the underlying soil. The characteristic differences were found during the entire sampling period between May and September. RFLP analyses of cloned nifH PCR products also revealed characteristic patterns for each sample type. Among 42 nifH clones obtained from a forest litter library nine different RFLP patterns were found, and among 64 nifHclones obtained from forest soil libraries 13 different patterns were found. Only two of the patterns were found in both the litter and the soil, indicating that there were major differences between the nitrogen-fixing microbial populations. A sequence analysis of clones representing the 20 distinct patterns revealed that 19 of the patterns had a proteobacterial origin. All of the nifH sequences obtained from the Douglas fir forest litter localized in a distinct phylogenetic cluster characterized by the nifH sequences of members of the genera Rhizobium, Sinorhizobium, and Azospirillum. The nifH sequences obtained from soil were found in two additional clusters, one characterized by sequences of members of the genera Bradyrhizobium,Azorhizobium, Herbaspirillum, andThiobacillus and the other, represented by a singlenifH clone, located between the gram-positive bacteria and the cyanobacteria. Our results revealed the distinctness of the nitrogen-fixing microbial populations in litter and soil in a Douglas fir forest; the differences may be related to special requirements for degradation and mineralization processes in the plant litter.

nifH gene diversity in the bacterial community associated with the rhizosphere of Molinia coerulea, an oligonitrophilic perennial grass

“…Cluster A dominated in both root and soil fractions (56% of all sequences, Table 1). Because of this dominance, the diversity of nifH gene pools was low as compared to other studies (Ueda et al, 1995;Widmer et al, 1999). No sequence in cluster A was related to so far characterized NifH sequences from cultivated organisms.…”

“…(Steenhoudt and Vanderleyden, 2000) were often described as being associated with grass roots. However, we did not detect any sequence related to these bacteria, even if the nifH genes from these organisms should have been amplified with the primers used (Widmer et al, 1999).…”

“…Representative samples (0.5 g) of fresh root and soil material were subjected to DNA extraction and purification using the bead-beating technique (Borneman et al, 1996). A nested-PCR amplification with consensus primers (Widmer et al, 1999) generated a 370 bp nifH gene fragment. PCR products were cloned into pGEM-T vector (Promega corp., Madison, WI).…”

“…Such approaches may be applied to functional genes, such as dinitrogen-fixation genes. The nifH gene was widely used to detect nitrogen-fixing bacteria (NFB) (Zehr and McReynolds, 1989;Ueda et al, 1995;Ohkuma et al, 1999;Piceno et al, 1999;Widmer et al, 1999;Zani et al, 2000;Poly et al, 2001). It encodes for the dinitrogenase reductase, a key enzyme in the nitrogen fixation process.…”

Endophytic nitrogen fixation in dune grasses (Ammophila arenaria and Elymus mollis) from Oregon

“…These bacteria may not be cultured by the methods used or may require metabolic interactions with other species normally present in planta but absent in pure culture. Recent PCR-based studies have revealed the presence of many unidentified and uncultured diazotrophs in such diverse habitats as rice roots [28] and forest litter [29]. In the case of the kallar grass/Azoarcus symbiosis, it has not been possible to re-isolate the bacterium even when the bacterium has been applied to sterile plants [40].…”