Host-specific ecotype diversity of rhizoplane diazotrophs of the perennialglasswort Salicornia virginica and selected salt marsh grasses

The degree of host specificity of most plant root associated bacteria is po orly understood. In this study we examined the physiological diversity of o xygen utilizing, culturable diazotrophs from the rhizoplane of the high mar sh perennial glasswort Salicornia virginica and compared them to diazotroph s from other salt marsh plants (tall and short Spartina alterniflora, Juncu s roemerianus, and Spartina patens) from the same ecosystem. Forty-six pure culture strains were recovered from the rhizoplane of S. virginica by stab inoculating freshly collected roots into combined nitrogen-free semi-solid media, followed by streak plating of clonal outgrowths. The majority of th ese strains were Gram-negative obligately aerobic or microaerophilic rods, but 3 Gram-positive strains were also isolated and characterized. API 20NE test strips were used for preliminary characterization of all strains, yiel ding 22 physiologically different API strain groups. One representative str ain was selected from each API group and tested for the presence of nifH, d enoting strains capable of Nz-fixation. Seventeen strains (14 Gram-negative , 3 Gram-positive) were nifH-positive and were characterized further using BIOLOG test plates. Four well-supported strain clusters were identified by bootstrapped cluster analysis of the BIOLOG substrate utilization profiles. These clusters differed in utilization of carbohydrates, carboxylic acids, and amino acids. S. virginica diazotrophs were physiologically quite diffe rent from rhizoplane diazotrophs from the low marsh plants S, alterniflora and J. roemenianus, but much more similar to diazotrophs from another high marsh plant, S. patens. We hypothesize that the observed physiological diff erentiation between high marsh and low marsh diazotrophs reflects differenc es in selection pressures in the rhizoplane microenvironment produced by pl ants with differing abilities to ventilate the rhizosphere. In addition, hi gh and low marsh branches were further resolved into host-specific strain c lusters, which also implies a strong impact of other host features, such as the suite of carbon exudate compounds produced, on the distributions of sp ecific diazotroph strains. These findings imply endemic, host-specific dist ributions of salt marsh diazotrophs and are consistent with the great diver sity of diazotrophs that have been observed in this ecosystem to date.