Several tropical grasses harbor symbiotic nitrogen-fixing bacteria within their stem and rhizome tissue that may contribute to the nitrogen nutrition of the host plant. We present evidence here that sand dune grasses (Ammophila arenaria and Elymus mollis) from Oregon also contain nitrogen-fixing bacteria. Surface-sterilized stem and rhizome tissue from these species possess acetylene reduction (nitrogen fixation) activity and large populations (10(5) to 10(6) cfu/g fresh weight) of bacteria. These bacteria were cultured on N-free media and identified by sequencing of 16S rRNA genes or by GC-FAME. Random sequencing of numerous colonies from the initial isolation plates of mixed isolates showed that pseudomonads (Stenotrophomonas and Pseudomonas) were by far the most common microorganism. One isolate -Burkholderia sp. strain Aa1 - reduced acetylene in culture with maximum activity at an O(2) concentration of 2% (v/v) in liquid media or 10% on solid media. PCR screening of all the isolates with nifH and nifD primers was positive only for this species. Immunolocalization studies with antibodies to nitrogenase resulted in labeling within plant cell walls of stems and rhizomes. Evidence for a similar nitrogen-fixing association was also detected in Uniola paniculata (sea oats) and Ammophila brevigulata (American beachgrass). We conclude that these grasses, and probably other dune grasses from temperate climates, contain endophytic, diazotrophic bacteria that may contribute to the phenomenal success of these grasses on nutrient-poor sand.