Diversity of nitrogen-fixing bacteria associated with switchgrass in the native tallgrass prairie of northern Oklahoma

Abstract

Switchgrass (Panicum virgatum L.) is a perennial C4 grass native to North America that is being developed as a feedstock for cellulosic ethanol production. Industrial nitrogen fertilizers enhance switchgrass biomass production but add to production and environmental costs. A potential sustainable alternative source of nitrogen is biological nitrogen fixation. As a step in this direction, we studied the diversity of nitrogen-fixing bacteria (NFB) associated with native switchgrass plants from the tallgrass prairie of northern Oklahoma (United States), using a culture-independent approach. DNA sequences from the nitrogenase structural gene, nifH, revealed over 20 putative diazotrophs from the alpha-, beta-, delta-, and gammaproteobacteria and the firmicutes associated with roots and shoots of switchgrass. Alphaproteobacteria, especially rhizobia, predominated. Sequences derived from nifH RNA indicated expression of this gene in several bacteria of the alpha-, beta-, delta-, and gammaproteobacterial groups associated with roots. Prominent among these were Rhizobium and Methylobacterium species of the alphaproteobacteria, Burkholderia and Azoarcus species of the betaproteobacteria, and Desulfuromonas and Geobacter species of the deltaproteobacteria.

FIG 1

Venn diagram showing the number of shared and unique OTUs among the three sample types (root DNA, root RNA, and shoot DNA). Numbers indicated in the diagram are OTU identity numbers (details of each OTU are given in Table 1).

FIG 2

Class-level composition of nifH clone libraries. Percent abundances per clone library from root RNA (242 sequences), root DNA (451 sequences), shoot DNA (397 sequences), and overall sequences (1,060) are shown.

FIG 3

Phylogeny and composition of nifH phylotypes representing four or more sequences. OTUs from this study are represented by OTU number and the associated protein accession number. The evolutionary history was inferred by using the maximum likelihood method based on the Poisson correction model. The bootstrap consensus tree was inferred from 1,000 replicates. There were a total of 113 positions in the final data set, and analyses were conducted in MEGA5. Sequences from known bacteria are indicated by name and NCBI protein accession numbers. This tree was rooted with the nifH gene from the archaeon Methanothermococcus okinawensis.